Method for recombinant production of antigen non-specific glycosylation inhibiting factor (GIF)

ABSTRACT

Polypeptides, polynucleotides, fragments thereof, and monoclonal antibodies thereto are provided for antigen-specific and antigen-non-specific glycosylation inhibiting factor and a method for recombinant production of biologically active polypeptides from a structural gene encoding the polypeptide.

This invention was made with Government support under grant numbersAI11202, AI14784, and AI32834 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

This is a continuation of application Ser. No. 08/061,041, filed May 14,1993, now abandoned, which is a continuation-in-part of PCT/US92/04614,filed Jun. 3, 1992, which is a continuation-in-part of Ser. No. 709,375,filed Jun. 3, 1991, now abandoned, which is a continuation-in-part ofSer. No. 533,889, filed Jun. 4, 1990 now abandoned.

FIELD OF THE INVENTION

The invention relates to human glycosylation inhibiting factor (GIF)which can be used to suppress the human immune response to an antigenand polynucleotide sequences encoding GIF.

DESCRIPTION OF THE BACKGROUND ART

Although the immune response is often seen as beneficial, in certaincircumstances the immune response to an antigen can actually be harmfulto the animal in which the immune response occurs. An example where theimmune response creates a condition wherein the host is subject toserious pathologic sequelae is in such autoimmune diseases as lupuserythematosus. In lupus erythematosus, antibodies are often presentwhich react with determinants in the thyroid, erythrocytes, DNA, andplatelets of the host.

Another example of where the suppression from immune response would bedescribed is in the treatment of allergies. It has been established thatIgE antibodies against allergens cause hay fever, and are involved inthe other allergic diseases such as extrinsic asthma. The crucial roleof IgE antibodies in the allergic diseases raised the possibility thatthe regulation and suppression of the IgE antibody formation againstallergens would be one of the fundamental treatments for allergicdiseases. For example, in the serum of hay fever patients sensitive toragweed allergens, IgE antibodies against the allergens are alwaysdetected. The IgE antibody titer goes up after the pollen season, andthen declines only slightly during the rest of the year. Since the halflife of IgE in the serum is only 2 to 3 days, the persistence of the IgEantibody titer indicates that the antibodies are being synthesizedcontinuously by the lymphoid cells of the patients in spite of the lackof allergen exposure.

Over the past 20 years, several different attempts were made to controlthe IgE antibody response in experimental animals. One of the approacheswas to improve classical immunotherapy or desensitization treatment, inwhich allergic patients receive repeated injections of a minute dose ofallergen. It was shown that the desensitization treatment can improveclinical symptoms in some patients. However, the IgE antibody titer inthe serum of hay fever patients did not decline after the treatment. Themajor immunological effects of the treatment is an enhancement of theIgG antibody formation, and the suppression of an increase in the IgEantibody titer after the pollen season.

A limitation in the desensitization, or immunosuppression treatment isthat patients cannot tolerate a large dose of allergen because of sideeffects. In order to overcome this difficulty, attempts were made to usea chemically modified allergen, such as urea-denatured antigen orpolyethylene glycol (PEG)-conjugates of the antigen for the treatment.Since the modified antigens do not bind to antibodies against the nativeantigen, relatively large doses of the modified antigen can be injectedwithout causing allergic symptoms. However, the modified antigen canstimulate antigen-specific T-cells. Evidence was obtained thatintravenous injections of the modified antigen into mice resulted in thegeneration of antigen-specific suppressor T-cells which suppressed theprimary IgE antibody response to the native antigen. However, thetreatment had minimal effects on the on-going IgE antibody formation, ifthe treatment were initiated after the antibody titer reached maximum(Takatsu and Ishizaka, J. Immunol., 117: 1211, 1976). In agreement withthe observations in the mouse, clinical trials ofpolyethylene-glycol-conjugated allergen in hay fever patients showedthat the treatment failed to diminish the IgE antibody titer. Failure ofthe repeated injections of the modified antigen to suppress the on-goingIgE antibody formation is probably due to the presence of a relativelylarge population of antigen-specific helper T-cells in the allergicpatients. Since the modified antigen not only induces the generation ofantigen-specific suppressor T-cells, but also expands the population ofhelper T-cells, this latter effect of the treatment might have overcomethe effect of suppressor T-cells. This interpretation is supported bythe fact that transfer of antigen-specific suppressor T-cells intoimmunized mice resulted in the suppression of the on-going IgE antibodyformation (Takatsu and Ishizaka, J. Immunol., 117: 1211, 1976). Theresults collectively suggested that the persistent IgE antibodyformation in hay fever patients could be suppressed, if it were possibleto generate the antigen-specific suppressor T-cells without expandingthe helper T-cell populations.

Since 1980, the inventors have investigated various ways in which IgEsynthesis is selectively regulated in an immunoglobulin isotype-specificmanner. As a result of this research, two types of T-cell factors havebeen found which have affinity for IgE and selectively regulate IgEsynthesis. One of the IgE-binding factors (IgE-BF) selectively enhancesthe IgE response, while the other type of IgE-BF selectively suppressesthe response. The major difference between the IgE-potentiating factorsand IgE-suppressive factors appears to be carbohydrate moieties in themolecules. The IgE-potentiating factors bind to lentil lectin andconcanavalin A, while IgE-suppressive factors fail to bind to theselectins (Yodoi, et al., J. Immunol., 128: 289, 1982). Analysis of thecellular mechanism for the selective formation of eitherIgE-potentiating factors or IgE-suppressive factors, as well as genecloning of the factors, indicated that the IgE-potentiating factor andIgE-suppressive factor share a common structural gene and that thenature of the carbohydrate moieties and biologic activities of thefactors are established during the post-translational glycosylationprocess (Martens, et al, Proc. Nat'l Acad. Sci., U.S.A., 84: 809, 1987).Under the physiological conditions, this glycosylation process iscontrolled by two T-cell factors which either enhance or inhibit thisprocess. These factors are denominated glycosylation inhibiting factor(GIF) and glycosylation enhancing factor (GEF).

A unique property of GIF is its biochemical activity. This lymphokinebinds to monoclonal antibodies against lipomodulin (a phospholipaseinhibitory protein) and appears to be a phosphorylated derivative of aphospholipase inhibitory protein (Uede, et al., J. Immunol, 130: 878,1983). It was also found in the mouse that the major source of GIF isantigen-specific suppressor T-cells (Ts) (Jadieu, et al., J. Immunol.,133: 3266, 1984). Subsequent experiments on ovalbumin (OVA)-specificsuppressor T-cell hybridomas indicated that stimulation of the hybridomacells with antigen (OVA)-pulsed syngeneic macrophages resulted in theformation of GIF that has affinity for OVA (antigen-binding GIF).However, the same hybridomas constitutively secreted GIF having noaffinity for OVA (nonspecific GIF). Studies on the relationship betweennonspecific GIF and OVA-binding GIF indicated that the antigen-bindingGIF is composed of an antigen-binding polypeptide chain and anonspecific GIF (Jardieu, and Ishizaka, in Immune Regulation ByCharacterized Polypeptides, Goldstein, et al., eds., Alan R. Liss, Inc.,N.Y., p595, 1987). It was also found that the antigen-binding GIF sharescommon antigenic determinants with antigen-specific suppressor T-cellfactors (TsF) described by the other investigators, and suppressed theantibody response in an antigen (carrier)-specific manner. Furthermore,not only antigen-binding GIF, but also antigen-specific TsF described byother investigators, bound to immunosorbent coupled with monoclonalanti-lipomodulin (141 -B9), and were recovered by elution of theimmunosorbent at acid pH.

Despite the major limitations of desensitization in treating allergy,this technique continues to be the method of choice. Consequently, thereis significant need for a technique which is antigen-specific yet doesnot have associated with it the side effects seen with existingdesensitization regimens.

The suppression of the immune response is crucial in order to preventhost versus graft (HVG) and graft versus host rejection (GVH).Unfortunately, in the case of both autoimmune disease as well as in HVGand GVH, the immune response suppression uses highly toxic drugs whichare of limited effectiveness and act systemically, rather thanspecifically. The severe limitations of such therapy point to the needfor immunosuppressive agents which have less toxicity, but greaterspecificity.

An improved way to suppress an immune response to an antigen in a humanwould be to administer an immunosuppressively effective amount of humanGIF which can specifically bind to the antigen. In so doing, theconcentration of T suppressor factor is favored and, as a result, theimmune response to the antigen is decreased. The present inventionprovides a means for accomplishing this result.

SUMMARY OF THE INVENTION

The invention provides substantially purified human antigen-specific andantigen non-specific GIF and the nucleotide sequence encoding GIF. Ageneral method for the recombinant production of biologically activepolypeptides is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and deduced amino acid sequence of a cDNAclone for murine GIF.

FIG. 2 shows the nucleotide and deduced amino acid sequence of a cDNAclone for human GIF.

FIG. 3 shows a map of the pST811 vector.

FIG. 4 shows a map of the pTMK-hGIF vector.

FIG. 5 shows a map of the SRα-hGIF vector.

FIG. 6 shows a map of the SRα-hcGIF vector.

FIG. 7 shows a map of the fusion expression vector, pMEproCT.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to substantially pure humanantigen-specific GIF with specificity for an antigen associated with anundesirable immune response. This human antigen-specific GIF is highlyuseful for the immunosuppression of the undesirable immune response inan antigen-specific manner.

Preferred in the present invention are human antigen-specific GIFs whichcan specifically bind allergens. In an especially preferred embodimentof the invention, a human antigen-specific GIF is disclosed which bindsto an epitope on bee venom phospholipase A₂ (PLA₂), the major allergenin honey bee venom. This specificity enables this antigen-specific GIF,and like antigen-specific GIFs with the same specificity, to be used tosuppress the human immune response to PLA₂. In another especiallypreferred embodiment of the invention, a human antigen-specific GIF isdisclosed which binds to an epitope on Japanese cedar pollen which canbe used to suppress the human immune response to this antigen.

The teaching used to produce GIF with antigen-specificity to bee venomPLA₂ can be readily extended to other antigens by those of skill in theart to prepare and purify other GIF molecules with antigenic specificityfor those other antigens without undue experimentation. As aconsequence, the broad pioneering nature of the invention enables thepreparation of human antigen-specific GIFs for other allergens which canbe used to suppress such immune response mediated disorders asautoimmune disease and allergy. The production of various humanantigen-specific GIFs is especially facilitated where the antigen of theundesirable immune response is known such as with most allergies andvarious autoimmune diseases.

The human PLA₂ -specific GIF of the invention is obtained from, or hasthe identifying characteristics of, an antigen-specific GIF obtainedfrom the cell line AC5 having ATCC accession number HB 10473. The humanJapanese cedar pollen-specific GIF of the invention is obtained from, orhas the identifying characteristics of, an antigen-specific GIF obtainedfrom the cell line 31E9 (ATCC HB 11052).

Methods of Producing and Characterizing Hybridomas

The general method used for production of hybridomas is well known(Kohler, et al., European J. Imm., 6: 292, 1976). Briefly, peripheralblood mononuclear cells (PBMC) from a human allergic to honey bee venomwere cultured in the presence of chemically modified PLA₂. Non-adherentcells were recovered and then cultured with IL-2 and lipocortin-1 priorto fusion with lymphoblastoid cell line BUC. Hybridomas were screenedfor production of human GIF specific for PLA₂.

More generally, the invention is directed to a method of producing acontinuous hybridoma cell line which produces human antigen-specific GIFcomprising:

(a) obtaining human antigen-primed T-cells which are activated to theantigen and cultured in the presence of IL-2 and a phospholipase A₂inhibitor; and

(b) combining the activated T-cells by fusion with a fusion partner cellline to produce hybridomas capable of producing human antigen-specificGIF.

The antigen-primed T-cells can be obtained from any sample, includingthe mononuclear cell fraction of peripheral blood. The antigen-primedT-cells can then be activated by culturing in the presence of theantigen to which they have been primed, followed by expanding theactivated T-cells in the presence of interleukin-2 (IL-2) and aphospholipase A₂ inhibitor. An especially useful phospholipase A₂inhibitor for such purposes is lipocortin. Alternatively, syntheticcompounds with PLA₂ inhibitory activity can be used such as2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, (ONO-RS-082, ONOPharmaceutical Co.).

Under certain circumstances, such as where the primary antigen is toxicto the T-cells, it is desirable to chemically modify the antigen. Agentsuseful for such modification include guanidine HCl and cyanogen bromide,but those of skill in the art can easily ascertain similar agentswithout undue experimentation. Generally, it is preferred to use agentswhich do not destroy the external structure of the antigen, since it isthought that such external structures are important in suppressor T-cellepitopic recognition of the antigen. However, this issue is notsignificant for most antigens, such as many allergens, which are notcytotoxic. Consequently, with typical allergens, the native moleculescan be used to stimulate the T-cells.

The present invention is directed to a method for generatingantigen-specific human T-cells and T-cell hybridomas which produce humanantigen-specific GIF, which are specifically reactive with an antigenwhich is associated with an immune response to be immunosuppressed.

The isolation of T-cell hybridomas producing a human antigen-specificGIF with the antigenic specificity of the human antigen-specific GIF ofthe invention can be accomplished using routine screening techniques todetermine the elementary reaction pattern of the human antigen-specificGIF of interest. Thus, for example in the case of human GIF specific forPLA₂, if a human antigen-specific GIF being tested suppresses the immuneresponse of cells from a patient allergic to PLA₂, then the humanantigen-specific GIF being tested and the human GIF specific for PLA₂produced by the hybridoma of the invention are equivalent.

Still another way to determine whether a human antigen-specific GIF hasthe specificity of a human antigen-specific GIF of the invention is topre-incubate the human antigen-specific GIF of the invention with theantigen with which it is normally reactive (for example, bee venomPLA₂), and determine if the human antigen-specific GIF being tested isinhibited in its ability to bind the antigen. If the humanantigen-specific GIF being tested is inhibited then, in all likelihood,it has the same epitopic specificity as the human antigen-specific GIFof the invention.

As used in this invention, the term "epitope" is meant to include anydeterminant capable of specific interaction with a humanantigen-specific GIF or the monoclonal antibodies of the invention.Epitopic determinants usually consist of chemically active groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

In still another aspect, the invention relates to a method of producingsubstantially pure human antigen-specific GIF comprising:

(a) culturing a continuous hybridoma cell line capable of producinghuman antigen-specific GIF such that the cell line produces humanantigen-specific GIF; and

(b) isolating substantially pure human antigen-specific GIF from theculture.

The continuous hybridoma cell lines so used can themselves be producedas described above. In addition, during culturing the hybridoma cellline is preferably stimulated to produce human antigen-specific GIF byexposing the hybridoma cells to syngeneic macrophages which have beenpulsed with the antigen to which the antigen-specific GIF binds, or withantibodies to the CD3 or T-cell receptor.

Various techniques can be used to isolate or substantially purify thehuman antigen-specific GIF from the culture. A particularly usefultechnique is affinity purification using the antigen, for exampleattached to a solid phase, to which the antigen-specific GIF binds. Amodification of this technique is to use two affinity absorption steps,if desirable, to substantially purify the human antigen-specific GIF. Insuch a process, the step of isolating substantially pure humanantigen-specific GIF includes:

(i) reacting the hybridoma cell line culture with a monoclonal antibodyspecifically reactive with human GIF;

(ii) eluting the human GIF from the monoclonal antibody;

(iii) reacting the eluted GIF with the antigen to which the humanantigen-specific GIF binds;

(iv) eluting the human antigen-specific GIF from the antigen; and

(v) recovering the human antigen-specific GIF.

Alternatively, immunosorbent coupled to the monoclonal antibody againstantigen-specific GIF such as 110BH3, may be used instead ofantigen-coupled immunosorbent. The step of isolating substantially purehuman antigen-specific GIF includes:

(i) reacting the hybridoma cell line culture with a monoclonal antibodyto which the human antigen-specific GIF binds;

(ii) eluting the human GIF from the monoclonal antibody;

(iii) reacting the eluted GIF with a monoclonal antibody specificallyreactive with human GIF;

(iv) eluting the human antigen-specific GIF from the monoclonalantibody; and

(v) recovering the human antigen-specific GIF.

Purification of human antigen-specific GIF is facilitated by adjustingthe hybridoma cells to serum-free culture medium, such as ABC. Aftertreatment of the subclone with anti-CD3, followed by culture of thehybridoma cells in Protein A-coated tissue culture dishes,antigen-binding GIF in culture supernatants can be purified byion-exchange chromatography, described above. Under such conditions, theprocess of isolating substantially pure human antigen-specific GIFincludes:

(i) contacting the hybridoma cell line culture supernatant with ananionic exchange matrix;

(ii) eluting the human GIF from the matrix;

(iii) reacting the eluted GIF with a monoclonal antibody specificallyreactive with human GIF or with the antigen to which the humanantigen-specific GIF binds, or both;

(iv) eluting the human antigen-specific GIF; and

(v) recovering the human antigen-specific GIF.

Thus, ion-exchange chromatographic purification is used in combinationwith affinity-purification to isolate human antigen-specific GIF, forexample, by using the antigen to which the antigen-specific GIF binds orby using an antibody specifically reactive with human GIF, or both, asdescribed above.

In the preferred embodiment, DEAE (diethylaminoethyl) Sepharose is thematrix utilized for purification of antigen-specific human GIF. Otherion-exchange materials which can be utilized include virtually any ofthe commercially available anion exchange agaroses and celluloses, suchas polysulfated agaroses, specifically including but not limited to QAE(quaternary amine) derivatives, ecteola(epichlorohydrintri-ethanolamine), TEAE (triethylaminoethyl)derivatives, and AE (aminoethyl) cellulose. The specific parameters forbinding and eluting from these various ion-exchange materials can beknown to those of skill in the art, or can be readily ascertained,without undue experimentation.

When the hybridoma cell line culture supernatant is added to theanion-exchange matrix equilibrated with about 20 mM salt, e.g., NaCl,much of the GIF will pass through the column and the remainder areeluted with salt concentrations up to about 60 mM. Preferred for elutionfrom DEAE are concentrations of NaCl from about 20 mM to about 60 mMcontained in 10 mM Tris.

A monoclonal antibody which is particularly useful in the affinitypurification of human GIF is the monoclonal antibody produced by a cellline 388F₁ or monoclonal antibodies having the specificity of amonoclonal antibody produced by cell line 388F₁ and a monoclonalantibody produced by a cell line 110BH3 or monoclonal antibodies havingthe same specificity.

THERAPEUTIC USES OF HUMAN ANTIGEN-SPECIFIC AND ANTIGEN NON-SPECIFIC GIF

The term "suppressive" denotes a lessening of the detrimental effect ofthe undesirable immune response in the human receiving therapy. The term"immunosuppressively effective" means that the amount of humanantigen-specific or non-specific GIF used is of sufficient quantity tosuppress the cause of disease or symptoms due to the undesirable immuneresponse.

The dosage ranges for the administration of the human GIF of theinvention are those large enough to produce the desired effect in whichthe symptoms of the immune response show some degree of suppression. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any counterindications. Dosage can vary from about 0.001mg/kg/dose to about 2 mg/kg/dose, preferably about 0.001 mg/kg/dose toabout 0.2 mg/kg/dose, in one or more dose administrations daily, for oneor several days.

The human GIF of the invention can be administered parenterally byinjection or by gradual perfusion over time. The human GIF of theinvention can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the human antigen specific andantigen non-specific GIF of the invention, the medicament being used fortherapy of an undesirable immune response to an antigen wherein theantigen is capable of binding by the human GIF of the invention.

The present invention is also directed to monoclonal antibodies, andB-cell hybridomas which produce them, which are specifically reactivewith human GIF. In addition, the invention provides monoclonalantibodies (and B-Cell hybridomas) which are specifically reactive withantigen-specific GIF but not with nonspecific GIF. A representativemonoclonal antibody of this type is 110BH3.

As stated above, techniques for production of hybridomas are well knownto those of skill in the art. In brief, the B-cell hybridomas of theinvention were prepared by immunizing BALB/c mice with affinity-purifiedhuman GIF and later boosted. Two weeks after the last immunization,spleen cells were obtained from the animals and transferred to syngeneicBALB/c mice which had been lethally irradiated. The syngeneic recipientswere immunized twice with purified human GIF and 2 weeks after the lastimmunization the spleen cells were fused with SP 2/0-14AG myeloma cellline. Hybridomas were screened for monoclonal antibody production tohuman GIF.

The isolation of hybridomas producing monoclonal antibodies with thereactivity of the monoclonal antibodies of the invention can beaccomplished using routine screening techniques to determine theelementary reaction pattern of the monoclonal antibody of interest.Thus, if a monoclonal antibody being tested reacts with human GIF, butdoes not react with mouse GIF, then the antibody being tested and theantibody produced by the hybridomas of the invention are equivalent.

The isolation of other hybridomas producing monoclonal antibodies withthe specificity of monoclonal antibody 388F₁, or any other monoclonalantibody of the invention, can be accomplished by one of ordinary skillin the art by producing anti-idiotypic antibodies (Herlyn, et al.,Science, 232: 100, 1986). An anti-idiotypic antibody is an antibodywhich recognizes unique determinants present on the monoclonal antibodyproduced by the hybridoma of interest. These determinants are located inthe hypervariable region of the antibody. It is this region which bindsto a given epitope and, thus, is responsible for the specificity of theantibody. The anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The animal immunizedwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies produced by thesecond animal, which are specific for the monoclonal antibodies producedby a single hybridoma which was used to immunize the second animal, itis now possible to identify other clones with the same idiotype as theantibody of the hybridoma used for immunization and thereby greatlysimplify and reduce the amount of screening needed to find otherhybridomas producing monoclonal antibodies with the specificity of themonoclonal antibodies of the invention.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

Alternatively, it is possible to evaluate, without undueexperimentation, a monoclonal antibody to determine whether it has thesame specificity as a monoclonal antibody of the invention bydetermining whether the monoclonal antibody being tested prevents themonoclonal antibody of the invention from binding to a particularantigen, for example human GIF, with which 388F₁ is normally reactive.If the monoclonal antibody being tested competes with 388F₁, forexample, as shown by a decrease in binding by 388F₁, then it is likelythat the two monoclonal antibodies bind to the same epitope. The similartest can be utilized for monoclonal antibody 110BH3.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention, such as 388F₁, isto pre-incubate 388F₁ with an antigen with which it is normallyreactive, for example, human GIF, and determine if the monoclonalantibody being tested is inhibited in its ability to find the antigen.If the monoclonal antibody being tested is inhibited then, in alllikelihood, it has the same epitopic specificity as the monoclonalantibody of the invention.

Under certain circumstances, monoclonal antibodies of one isotype mightbe more preferable than those of another in terms of their diagnostic ortherapeutic efficacy. Particular isotypes of a monoclonal antibody canbe prepared either directly, by selecting from the initial fusion, orprepared secondarily, from a parental hybridoma producing monoclonalantibody of different isotype, by using the sib selection technique toisolate class-switch variants (Steplewski, et al., Proceedings ofNational Academy of Sciences, USA, 82: 888653, 1985; Spira, et al.,Journal of Immunological Methods, 74: 307, 1984). Thus, the monoclonalantibodies of the invention would include class-switch variants havingthe specificity of monoclonal antibody 388F₁ which is produced by ATCCHB 10472. This cell line was placed on deposit for 30 years at theAmerican Type Culture Collection (ATCC) in Rockville, Md. prior to Jun.4, 1990.

The term "antibody" as used in this invention is meant to include intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab')₂, which are capable of binding the epitopic determinant.

The monoclonal antibodies of the invention can also be used inimmunoaffinity chromatography for the purification of the various typesof human GIF mentioned herein. One way by which such immunoaffinitychromatography can be utilized is through the use of, for example, thebinding of the monoclonal antibodies of the invention toCNBr-Sepharose-4B, Affigel (BioRad), or Tresyl-activated Sepharose(Pharmacia). These solid phase-bound monoclonal antibodies can then beused to specifically bind human GIF from mixtures of other proteins toenable its isolation and purification. The bound GIF can be eluted fromthe affinity chromatographic material using techniques known to those ofordinary skill in the art such as, for example, chaotropic agents, lowpH, or urea.

In another embodiment, the invention provides a substantially purefusion polypeptide R₁ - X₁ -X₂ -X₁ -X₂ -Lys-Arg!-R₂, wherein R₁ is acarrier peptide, R₂ is a polypeptide encoded by a structural gene, X₁ isLys or Arg, and X₂ is any amino acid. The "carrier peptide", or signalsequence, is located at the amino terminal end of the fusion peptidesequence. In the case of eukaryotes, the carrier peptide is believed tofunction to transport the fusion polypeptide across the endoplasmicreticulum. The secretory protein is then transported through the Golgiapparatus, into secretory vesicles and into the extracellular space or,preferably, the external environment. Carrier peptides which can beutilized according to the invention include pre-pro peptides whichcontain a proteolytic enzyme recognition site. Acceptable carrierpeptides include the amino terminal pro-region of calcitonin or otherhormones, which undergo cleavage at the flanking dibasic sites.Procalcitonin is processed by prohormone convertase which recognizesLys-Arg cleavage site. However, it should be noted that the invention isnot limited to the use of this peptide as a carrier. Other carrierpeptides with similar properties to pro-calcitonin described herein areknown to those skilled in the art, or can be readily ascertained withoutundue experimentation.

In one embodiment of the invention, a carrier peptide which is a signalsequence is included in the expression vector, specifically locatedadjacent to the N-terminal end of the carrier protein. This signalsequence allows the fusion protein to be directed toward the endoplasmicreticulum. Typically, the signal sequence consists of a leader of fromabout 16 to about 29 amino acids, starting with two or three polarresidues and continuing with a high content of hydrophobic amino acids;there is otherwise no detectable conservation of sequence known. Whilethe vector used in the example of the present invention uses thepro-region of calcitonin, other signal sequences which provide the meansfor transport of the fusion protein to the endoplasmic reticulum andinto the external environment, will be equally effective in theinvention. Such signal sequences are known to those of skill in the art.

The carrier peptide of the invention contains a proteolytic enzymerecognition site which has a dibasic motif (Lys-Arg) which contains anadditional Arg/Lys residue at the P4 and P6 positions. Differences inthe cleavage recognition site may imply that different processingenzymes exist for the proteolytic specificity. Preferably, the cleavagesite is about 6 amino acids having the sequence X₁ -X₂ -X₁ -X₂ -Lys-Arg,where X₁ is Lys or Arg and X₂ is any amino acid. This recognition siteallows for an unexpectedly high level of active protein encoded by thestructural gene to be produced.

Examples of processing enzymes which recognize the proteolytic siteinclude the mammalian enzyme, furin, the homologue of the yeastpropeptide processing enzyme Kex2, and other prohormone convertases(PCs). Preferably, the carrier peptide of the invention contains at thecleavage site within the precursor, a proteolytic enzyme recognitionsite, with a polynucleotide sequence encoding Arg/Lys-X₂ -Arg/Lys-X₂-Lys-Arg.

The fusion polypeptide of the invention includes a polypeptide encodedby a structural gene, preferably at the carboxy terminus of the fusionpolypetide. Any structural gene is expressed in conjunction with thecarrier and cleavage site. The structural gene is operably linked withthe carrier and cleavage site in an expression vector so that the fusionpolypeptide is expressed as a single unit. GIF is an example of astructural gene that can be used to produce a fusion polypeptide of theinvention.

The invention provides a substantially pure polypeptide. The term"substantially pure" as used herein refers to a polypeptide which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify the polypeptide using standard techniques for proteinpurification, such as affinity chromatography using a monoclonalantibody which binds an epitope of the polypeptide. The substantiallypure polypeptide will yield a single major band on a polyacrylamide gel.The purity of the polypeptide can also be determined by amino-terminalamino acid sequence analysis. The polypeptide includes functionalfragments of the polypeptide, as long as the activity of the polypeptideremains. Smaller peptides containing the biological activity ofpolypeptide are included in the invention.

The invention also provides polynucleotides encoding the fusionpolypeptide. These polynucleotides include DNA, cDNA and RNA sequences.It is understood that all polynucleotides encoding all or a portion ofthe fusion polypeptide are also included herein, as long as they encodea polypeptide of which the cleavage product has biological activity.Such polynucleotides include naturally occurring, synthetic, andintentionally manipulated polynucleotides. For example, thepolynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequence also includes antisense sequences and sequencesthat are degenerate as a result of the genetic code. There are 20natural amino acids, most of which are specified by more than one codon.Therefore, all degenerate nucleotide sequences are included in theinvention as long as the amino acid sequence of the fusion polypeptideencoded by the nucleotide sequence is functionally unchanged.

The invention also provides polynucleotides which are complementary tothe nucleotide sequences of the invention. A "complementary" nucleotidesequence will hybridize to a specific nucleotide sequence underconditions which allow the complementary sequence to hybridize. Theseconditions include temperature, pH, buffer and nucleotide composition.For example, the positive and negative strands of a double-stranded DNAmolecule are complementary nucleotide sequences. Polynucleotides of theinvention include fragments which are at least 15 bases in length, andtypically 18 bases or greater, which selectively hybridize to genomicDNA which encodes the polypeptide of interest. Selective hybridizationdenotes conditions (e.g., pH, temperature, buffer) which avoidnon-specific binding of a nucleotide sequence to the target DNA which isits complement.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences; 2) antibody screening of expression libraries todetect shared structural features; and 3) synthesis by the polymerasechain reaction (PCR).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9: 879, 1981).

An antigen non-specific GIF containing cDNA library, for example, can bescreened by injecting the various cDNAs into oocytes, allowingsufficient time for expression of the cDNA gene products to occur, andtesting for the presence of the desired cDNA expression product, forexample, by using antibody specific for antigen non-specific GIFpolypeptide or by using functional assays for GIF activity.Alternatively, a cDNA library can be screened indirectly for antigennon-specific GIF polypeptides having at least one epitope usingantibodies specific for antigen non-specific GIF polypeptides. Suchantibodies can be either polyclonally or monoclonally derived and usedto detect expression product indicative of the presence of antigennon-specific GIF cDNA.

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA.

The development of specific DNA sequences encoding a polypeptide canalso be obtained by: 1) isolation of double-stranded DNA sequences fromthe genomic DNA; 2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods for developingspecific DNA sequences for use in recombinant procedures, the isolationof genomic DNA isolates is the least common. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides due to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay et al., Nucl. Acid Res. 11: 2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for expression of a polypeptide having at least one epitope,using antibodies specific for the polypeptide. Such antibodies can beeither polyclonally or monoclonally derived and used to detectexpression product indicative of the presence of protein encoded by thecDNA.

DNA sequences encoding the fusion polypeptide of the invention can beexpressed in vitro by DNA transfer into a suitable host cell. "Hostcells" are cells in which a vector can be propagated and its DNAexpressed. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication.However, such progeny are included when the term "host cell" is used.Methods of stable transfer, in other words when the foreign DNA iscontinuously maintained in the host, are known in the art.

In the present invention, the polynucleotide sequences may be insertedinto a recombinant expression vector. The term "recombinant expressionvector" refers to a plasmid, virus or other vehicle known in the artthat has been manipulated by insertion or incorporation of the geneticsequences for antigen non-specific GIF, for example, and a carrierpeptide. Such expression vectors contain a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263: 3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding the polypeptide of the invention canbe expressed in either prokaryotes or eukaryotes. Hosts can includemicrobial, yeast, insect and mammalian organisms. The preferred host ofthe invention is a eukaryote. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. It is preferablethat the host cell of the invention naturally encodes an enzyme whichrecognizes the cleavage site of the fusion protein. However, if the hostcell in which expression of the fusion polypeptide is desired does notinherently possess an enzyme which recognizes the cleavage site, thegenetic sequence encoding such enzyme can be cotransfected to the hostcell along with the polynucleotide sequence for the fusion protein.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransfected with DNA sequences encoding the fusion polypeptide of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Techniques for the isolation and purification of either microbially oreukaryotically expressed polypeptides of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLE 1 PREPARATION OF HYBRIDOMA CELL LINES PRODUCING HUMANANTIGEN-SPECIFIC GLYCOSYLATION INHIBITING FACTOR (GIF) AND PURIFICATIONTECHNIQUES

A. ANTIGENS

Lyophilized phospholipase A₂ (PLA₂) from bee venom was purchased fromSigma Chemical Co., St. Louis, Mo. Denatured PLA₂ (-PLA₂) and cyanogenbromide-treated PLA₂ were prepared by the method described by King, etal., Arch. Biochem and Biophys., 172: 661, 1976. For the preparation ofD-PLA₂, 5 mg of PLA₂ were dissolved in 0.1M Tris HCl buffer, pH 8.6, anddenatured in 6M guanidine HCl in the presence of 5 mg/ml dithiothreitol.After 18 hours at room temperature, sulfhydryl groups werecarboxymethylated with iodoacetic acid. The denatured protein wasdialyzed against 0.02M acetic acid and kept at -40° C. until use. Forthe cleavage of methionine bonds in PLA₂, 10 mg bee venom PLA₂ wasdissolved in 0.4 ml distilled water, and 1.2 ml formic acid containing100 mg CNBr were added. After 2 hours at room temperature, the mixturewas diluted two-fold with H₂ O and lyophilized in Speed Vac. Native PLA₂was coupled to Tresyl activated Sepharose (Pharmacia) following theprocedures recommended by the manufacturer. Unless otherwise stated, 1mg protein was coupled to 1 ml Sepharose.

B. ANTIBODIES

Purified human E myeloma protein PS, monoclonal mouse IgE from thehybridoma H-1 DNP-E-26 (Liu, et al., J. Immunol., 124: 2728, 1980) andmonoclonal anti-CD3 (OKT 3) were the same preparations as thosedescribed in a previous article (Carini, et al., J. Immunol. Methods,127: 221, 1990). Ascitic fluid containing the monoclonal anti-T-cellreceptor αβ, WT 31C, (Spits, et al., J. Immunol., 135: 1922, 1985) waskindly supplied by Dr. J. DeVries, DNAX Institute of Molecular andCellular Biology, Palo Alto, Calif. The mouse monoclonal antibodyagainst rabbit lipomodulin 141B9 (Iwata, et al., J. Immunol., 132: 1286,1984) was the same preparation as that described in a previous article(Askasaki, et al., J. Immunol., 131: 3172, 1986). Specifically-purifiedgoat antibodies against mouse IgG, which contained both anti-heavy (γ)chain and anti-light chain, were previously described (Suemura, et al.J. Immunol, 125: 148, 1980). Fluoresceinated goat anti-mouse IgGantibodies were purchased from Cappel. Human IgE and anti-lipomodulinantibody (141B9) were coupled to CL-Sepharose 4B; approximately 5 mg ofprotein were coupled to 1 ml Sepharose.

C. CELL LINES

RPMI 8866 lymphoblastoid cells were cultured in RPMI 1640 mediumenriched with 10% fetal calf serum, 2 mM L-glutamine, 50 μM2-mercaptoethanol and antibiotics (RPMI 1640 culture medium). The mouseT-cell hybridoma 12H5 cells (Iwata, et al., J. Immunol., 140: 2534,1988) were maintained in high glucose Dulbecco's modified Eagle's medium(DMEM) described in a previous article (Huff, et al., Proc. Natl. Acad.Sci., USA, 129: 509, 1982). A hypoxanthine guaninephosphoribosyltransferase-deficient mutant of the human lymphoblastoidcell line CEM (BUC) cells were previously described (Huff & Ishizaka,Proc. Natl. Acad. Sci., USA, 81: 1514, 1984).

D. CELL CULTURE AND CONSTRUCTION OF HYBRIDOMAS

Peripheral blood was obtained from a patient allergic to honey beevenom, and mononuclear cells of the blood (PBMC) were obtained bycentrifugation on Ficoll-Paque (Pharmacia). To activate antigen-primedT-cells, PBMC were suspended in RPMI 1640 culture medium at theconcentration of 3×10⁶ nucleated cells/ml, and cultured for three daysin the presence of 10 μg/ml D-PLA₂ or CNBr-treated PLA₂. Non-adherentcells were recovered, resuspended in fresh culture medium (2×10⁵cells/ml), and then cultured for four days with 60 units/ml purifiedIL-2 (chromatographically purified human IL-2, Electro-nucleonics,Silver Spring, Md.), in the presence of 3 μg/ml recombinant humanlipocortin 1, which was kindly supplied by Drs. J. Browning and B.Pepinsky, Biogen.

To construct T-cell hybridomas, 1.2×10⁷ T-cells, which had beenpropagated by IL-2, were mixed with twice the number of BUC cells (asubline of CEM). Mixed cells were pelleted together and fused by usingpolyethylene glycol (1300-1600 MW., Sigma). Detailed procedures for cellfusion were as previously described (Huff, et al., Proc. Nat'l. Acad.Sci., U.S.A. 81: 1514, 1984). Cells were resuspended inhypoxanthine/aminopterin/thymidine (HAT)-containing DMEM, and 5×10⁴cells were seeded in each well of 96 well plates. Hybrid clones weremaintained in complete DMEM with biweekly subcultures. In order tostimulate the T-cell hybridomas, the cells were treated with 8 μg/ml OKT3 for 40 minutes at 0° C., and the antibody-treated cells (1×10⁶ /ml)were seeded in Limbro tissue culture wells (Flow Labs, McLean, Va.)which had been coated with 10 μg/ml anti-MGG. Culture supernatants wereobtained after 24 hour culture.

E. DETECTION OF CD3 AND TcR

The hybridoma cells (1×10⁶ /sample) were incubated 40 minutes at 0° C.with 8 μg/ml OKT 3 or a 1:1000 dilution of anti-TcRαβ(WT31)-containingascitic fluid in RPMI 1640 medium supplemented with 5% FCS and 10 μMNaN3. As controls, aliquots of the same cells were treated with the sameconcentration of mouse IgG_(2a) (Becton-Dickinson, isotype control).Cells were washed twice with PBS containing 5% FCS, and then incubatedwith fluoresceinated anti-mouse IgG for 40 minutes. After washings,cell-associated fluorescence was analyzed by using FACScan fromBecton-Dickinson.

The CD3⁺ hybridoma cells were identified by rosetting using oxerythrocytes coated with anti-mouse IgG. The antibodies were coupled toerythrocytes by the method of Wilhelm, et al., (J. Immunol. Methods, 90:89, 1986). Briefly, 0.5 ml of packed ox erythrocytes were washed 4 timeswith saline, and resuspended in 0.75 ml of 0.5 μg/ml purified anti-MGG;25 μl of CrCl₃ (16.5 mg CrCl₃ dissolved in 5 ml saline) were added tothe cell suspension under gentle mixing, and the cell suspension wasincubated for 1 hour at 30° C. The anti-MGG-coupled erythrocytes werewashed 4 times with saline and resuspended in 5 ml FCS (approximately1×10⁹ erythrocytes/ml). To detect the CD3⁺ cells, pellets of 10⁶hybridoma cells were suspended in 80 μl of DPBS containing 5% FCS and 8μg/ml OKT 3. After 45 minutes at 0° C., the cells were washed twice andresuspended in 80 μl DPBS-5% FCS, and 20 μl of a suspension ofanti-MGG-coated erythrocytes and crystal violet were added to the cellsuspension. The mixtures were centrifuged at 200 g for 5 minutes andtubes were incubated for 2 hours at 0° C. The pellets were gentlyresuspended and examined for rosetting cells under microscope.

F. ENRICHMENT OF CD3⁺ CELLS

Hybridoma cells treated with 8 μg/ml OKT 3 (1.5×10⁶ cells) were mixedwith anti-MGG coupled erythrocytes (ca 4×10⁸ erythrocytes) to formrosettes by the procedures described above. Pellets were resuspended andapplied to the top of Percoll gradient consisting of 60% and 50% Percolllayers. Tubes were centrifuged for 20 minutes at 1200 RPM (700 g) atroom temperature. The pelleted cells were washed twice with culturemedium, and the erythrocytes were lysed by treatment with 0.83% NH₄ Clbuffer for 1 minute at 0° C. The cells were washed with and resuspendedin DME culture medium and cultured to expand the cell population.

Further enrichment of CD3⁺ cells was carried out by cell sorting.Hybridoma cells were treated with OKT 3 and stained with fluoresceinatedanti-MGG. The positively stained cells were selected by sorting thecells by using FACSTAR (Becton-Dickinson).

G. PURIFICATION AND DETECTION OF IgE-BF

Culture supernatant of T-cell hybridomas were filtered through Diaflo YM100 membranes (Amicon Corp., Lexington, Mass.) and the filtrates wereconcentrated ten-fold by ultrafiltration through YM5 membranes. IgE-BFin the filtrates were purified by using IgE-coupled Sepharose by thedescribed procedures (Ishizaka & Sandberg, J. Immunol, 126: 1692, 1981).The presence of IgE-BF in culture filtrates or acid eluate fraction fromIgE-Sepharose was assessed by inhibition of rosette formation of FcεR⁺ Blymphoblastoid cell line, RPMI 8866 cells with human IgE-coated oxerythrocytes (E-IgE) by the procedures previously described (Kisaki, etal., J. Immunol., 138: 3345,1987). The proportion of rosette formingcells (RFC) in 300 RPMI 8866 cells was determined in triplicate and wasexpressed as the average ±SD.

Rodent IgE-BF formed by the 12H5 cells were detected by the sameprocedure, except that rat IgE-coated ox erythrocytes were employed asindicator cells, and mesenteric lymph node cells of Lewis strain rateinfected with Nipportronngylus brasiliensis were used as a source ofFcεR⁺ B cells (Yodoi & Ishizaka, J. Immunol, 124: 1322,1980).

H. DETECTION OF GIF

GIF was detected by using T-cell hybridoma 12H5 cells (Iwata, et al., J.Immunol., 140: 2534, 1988). A suspension of the hybridoma cells wasmixed with an equal volume of a test sample, and the cell suspensionswere cultured for 24 hours with 10 μg/ml mouse IgE. Culture supernatantswere filtered through CF50A membranes, and filtrates containing IgE-BFwere fractionated on lentil lectin Sepharose (Yodoi, et al., J.Immunol., 125: 1436, 1980). Both unbound proteins (effluent fraction)and those eluted with 0.2M α methylmannoside (eluate fraction) wereassessed for the presence of IgE-BF by rosette inhibition technique.When the 12H5 cells were cultured with mouse IgE alone, essentially allIgE-BF formed by the cells bound to lentil lectin Sepharose and wererecovered by elution with α methylmannoside. Thus, the ratio of thepercent rosette inhibition between the effluent/eluate fraction is lessthan 0.2. If a sufficient amount of GIF were added to the culture of12H5 cells together with mouse IgE, the majority of IgE-BF formed by thecells lacked affinity for lentil lectin and were recovered in theeffluent fraction (Iwata & Ishizaka, J. Immunol., 141: 3270, 1988).Thus, GIF was taken as (+), if the ratio of the percent rosetteinhibition between the effluent/eluate fraction were 3.0 or higher.

I. FRACTIONATION OF GIF

In order to determine whether GIF from hybridomas has affinity for beevenom PLA₂, culture filtrates of hybridoma cells were fractionated onantigen-coupled Sepharose. Hybridoma cells were treated with OKT 3antibody (8 μg/ml) and 8 ml aliquots of the antibody treated oruntreated cell suspension (1.5×10⁶ cells/ml) were cultured inanti-MGG-coated tissue culture flasks. Culture supernatants wereconcentrated four-fold, and a 2 ml sample was absorbed with 0.4 mlIgE-Sepharose. The effluent fraction was mixed with 0.5 ml PLA₂-Sepharose overnight, and immunosorbent was packed into a small column.After effluent fraction was recovered, the column was washed with DPBS,and then eluted with 1.0 ml glycine HCl buffer, pH 3.0. Partialpurification of GIF on anti-lipomodulin (141B9) Sepharose was carriedout by the procedures previously described (Akasaki, et al., J.Immunol., 136: 3172, 1987).

J. DETERMINATION OF PHOSPHOLIPASE INHIBITORY ACTIVITY

Affinity-purified GIF was treated with alkaline phosphatase aspreviously described (Uede, et al, J. Immunol, 139: 898, 1983). Briefly1 ml of the preparation was dialyzed against Tris-HCl buffer, pH 8.2 andwas mixed with 1 unit of insoluble alkaline phosphatase (calfintestinal, Sigma) for 2 hours at room temperature. Aftercentrifugation, the supernatant was dialyzed against 0.1M Tris-HClbuffer, pH 8.0. Phospholipase A₂ inhibitory activity of thealkaline-phosphatase treated samples was determined using E coli whichwere biosynthetically labeled with ³ H-oleic acid and porcine pancreaticPLA₂ (Sigma) (Rothut, et al., Biochem. Biophys. Res. Commun., 117: 878,1983). Detailed procedures were described in Ohno, et al. (Internat.Immunol., 1: 425, 1989). Briefly, porcine pancreatic PLA₂ (1×10⁻⁵ units)was mixed with GIF in a total volume of 150 μl. After 5 minutes at 25°C., 50 μl of a suspension of ³ H-labeled E coli (5000 cpm) was added,and the mixtures were incubated for 5 minutes at 25° C. The reaction wasstopped by the addition of 50 μl 2M HCl, and 50 μl of 100 mg/ml BSA wasadded to the mixtures. The suspensions were centrifuged for 1 minute at5500 g, and radioactivity in 250 μl of supernatant was measured in ascintillation spectrometer.

K. ION EXCHANGE COLUMN CHROMATOGRAPHY

Culture supernatant of AC5 cells in serum-free medium was concentrated25 to 100 fold by ultrafiltration. After centrifugation at 10,000 rpmfor 20 min, the supernatant was diluted 8-fold with distilled water,adjusted to pH 8.0 with Tris, and immediately applied to aDEAE-Sepharose CL-6B (Pharmacia) column (3 ml volume) which wasequilibrated with 10 mM Tris HCl buffer, pH 8.0. After effluent(passed-through) fraction was recovered, the column was washed with 4column volumes of 10 mM Tris-HCl buffer containing 20 mM NaCl, and thewashing was combined to the passed through fraction. Proteins bound tothe column were eluted successively with 4 column volumes of 10 mM TrisHCl buffer, pH 8.0 containing 50 mM, 75mM, 100 mM, 150 mM, and 200 mMNaCl. Each eluate fraction was concentrated and dialyzed againstDulbecco's phosphate buffered saline (DPBS).

L. GEL FILTRATION

One ml sample in DBPS was applied to a Superose 12 column (1.6×50 cm,Pharmacia), connected to HPLC (Beckman, System Gold). Proteins wereeluted from the column with DPBS at a flow rate of 1 ml/min, andappropriate fractions were collected. The column was calibrated withhuman IgE (PS protein, MW: 185,000), bovine serum albumin (BSA, MW:67,000), ovalbumin (MW: 43,000), soybean trypsin inhibitor (MW: 20,100),and cytochome C (MW: 12,500). All standard proteins except IgE wereobtained from Sigma. Retention time for the standard proteins were41.97, 52.08, 55.135, 62.097, and 71.67 min, respectively.

M. AFFINITY-PURIFICATION OF GIF

Culture supernatant of CL3 clone in complete DME medium was concentrated5-fold by ultrafiltration, and GIF in the supernatant was absorbed to141B9-Sepharose or anti-GIF Sepharose by recycling the supernatantovernight through the immunosorbent column (5 ml volume) (Iwata, et al.,J.Immunol., 141: 3270, 1988). The immunosorbent was washed with 20column volumes of DPBS, and proteins bound to the beads were recoveredby elution with 0.1M glycine HCl buffer, pH 3.0. Murine GIF from the 231F1 cells was purified by the same technique using the 141B9-Sepharose.

In order to isolate GIF in culture supernatant of AC5 cells inprotein-free medium, the supernatant was concentrated 50 to 100-fold byultrafiltration. An appropriate fraction of the supernatant from aDEAE-Sepharose column was concentrated to 5-6 ml and mixed overnight at4° C. with 1.0 to 1.5 ml of Affigel 10-immunosorbent coupled withmonoclonal anti-GIF antibody. The suspension was packed into a smallcolumn and the immunosorbent was washed with 40 column volumes of DPBS.In some experiments, the immunosorbent was washed with 40 column volumesof DPBS and 20 column volumes of PBS containing 0.5M NaCl. Proteinsbound to the immunosorbent were eluted with 0.05M glycine HCl buffercontaining 0.15M NaCl, pH 3.0-3.2.

N. DETECTION OF GIF BY SDS-PAGE

Affinity-purified GIF was dialyzed against 0.01% SDS in deionized water,and lyophilized in a Speed vac (Savant Instruments, Hicksville, N.Y.).Samples were then analyzed by SDS gel electrophoresis in 15%polyacrylamide slab gel by using the Laemmli system (Laemmli, U.K.,Nature, 227: 680, 1970). Gels were fixed and protein bands were detectedby silver staining (Ochs, et al., Electrophoresis, 2: 304, 1981).Molecular weight standards were obtained from Pharmacia.

O. ELISA ASSAYS

In order to detect monoclonal anti-GIF antibody, the method described bySteele, et al. (J.Immunol., 142: 2213, 1989) was employed with slightmodifications. Briefly, Immulon I plates (Dynatech) were coatedovernight with 100 μl of affinity-purified GIF diluted with 0.1Mcarbonate coating buffer, pH 9.6. Plates were washed 3 times withphosphate buffered saline (PBS) containing 0.05% Tween 20 between eachof the following steps. Plates were blocked with 2% BSA in PBS for 6-9hours. One hundred microliters of each test sample was then added to thewell, and plates were kept overnight at 4° C. Binding of mouse Ig to theplate was detected by using alkaline phosphatase-coupled goat anti-mouseIg (Zymed Lab, So. San Francisco, Calif.) and alkaline phosphatasesubstrate (Sigma). ELISA signal was read in a microplate reader MR 5000(Dynatech Lab) with a 410 nm filter 30 min after the addition ofsubstrate. Isotype of monoclonal antibodies was determined with ELISAassay by using an isotyping kit for mouse mAb (Zymed Lab).

For the detection of GIF in fractions of an affinity-purified GIFpreparation, a biotin-avidin system and amplification method (Stanley,et al., J.Immunol. Methods, 83: 89, 1985) were employed to increase thesensitivity. Maxi-Sorp microtiter plates (Nunc, Copenhagen, Denmark)were coated with 50 μl of each fraction. After incubation for 2 hours at37° C., plates were washed with Tween/PBS and blocked with 2% BSAovernight at 4° C. After washing, 50 μl of biotin-coupled mAb 141-B9(200 ng/ml) were added to each well and the plate was incubated for 2hours at 37° C. The plate was washed and 50 μl of a 1:1500 dilution ofstreptavidin-alkaline phosphatase conjugate (Zymed Lab) were added toeach well. After incubation for 1 hour at 37° C., quantities of alkalinephosphatase bound to the wells were measured by amplification system(Stanley, et al., J.Immunol. Methods, 83: 89, 1985), (GIBCO-BRL,Bethesda, Md.). ELISA signal was determined at 490 nm.

EXAMPLE 2 CHARACTERIZATION OF HYBRIDOMAS PRODUCING HUMANANTIGEN-SPECIFIC GIF

As described above, PBMC of a bee venom-sensitive patient were culturedfor three days in the presence of 10 μg/ml D-PLA₂, and activated T-cellswere propagated by IL-2 for four days in the presence of 3 μl/mlrecombinant lipocortin. T-cells were then fused with BUC cells toconstruct hybridomas. In this experiment, 4 hybridoma clones wereobtained. Each hybridoma clone was cultured in complete DMEM and culturesupernatants were filtered through YM100 membranes. Filtrates wereconcentrated ten-fold and assessed for the presence of GIF by using the12H5 cells. The results shown in Table I indicate that two of the fourhybridoma clones constitutively secrete GIF.

                  TABLE I                                                         ______________________________________                                        Selection of GIF-Producing Hybridomas                                                           .sup.3 H-Oleic Acid Release.sup.c                                      GIF Activity.sup.a                                                                         Release  Inhibition                                   Hybridoma  Effluent/Eluate                                                                            (cpm)    (%)                                          ______________________________________                                        Cl 1       0/26 (-)     ND       --                                           Cl 2       2/33 (-)     390 ± 27                                                                             4                                           Cl 3       29/0 (+)     257 ± 25                                                                            37                                           Cl 7       27/5 (+)     303 ± 17                                                                            26                                           Control    0/31.sup.b   408 ± 15                                                                            --                                           ______________________________________                                         .sup.a Culture filtrates of each clone were concentrated tenfold. One         volume of the filtrate was added to an equal volume of a suspension of th     12H5 cells, and the cells were cultured for 24 hours in the presence of 1     μg/ml mouse IgE. Numbers in the column represent the percent of rosett     inhibition by the effluent/eluate fractions from lentil lectin Sepharose.     The proportion of IgERFC in the absence of IgEBF was 24.4 ± 0.3 (SD)%.     .sup.b The 12H5 cells were cultured with 10 μg/ml mouse IgE alone, and     IgEBF in culture filtrates were fractionated on lentil lectin Sepharose.      .sup.c Culture filtrates were fractionated on 141B9Sepharose, and acid        eluates from the immunosorbent were concentrated to 1/100 volume of the       original culture supernatant. The samples were treated with alkaline          phosphatase, and dephosphorylated materials were assessed for the ability     to inhibit pancreatic phospholipase A2.                                  

The presence of CD3 determinants on the hybridoma clone CL3 was assessedby fluorocytometry and the rosetting technique. The cells were treatedwith 8 μg/ml monoclonal antibody OKT3 and then stained withfluoresceinated goat anti-mouse Ig. Less than 10% of the total cellswere stained. It was also found that only 6-8% of the OKT3-treated cellsformed rosettes with anti-MGG-coupled erythrocytes. As a consequence,the CD3⁺ cells were enriched using the rosetting procedures described inExample 1. Cells which formed rosettes with anti-MGG couplederythrocytes were separated from non-rosetting cells by density gradientcentrifugation on Percoll layers and were expanded by culture incomplete DMEM. The same procedures were repeated three times to enrichthe CD3⁺ cell population. Treatment of the final cell preparation withOKT3 antibody followed by incubation with anti-MGG-coated erythrocytesshowed that 80-90% of the cell population formed rosettes. Approximately75% of the cells were stained by OKT3 in cytofluorometry. However, whenculture of the cells for 2 weeks with four passages resulted in thedecline of CD3⁺ cells to approximately 52% (as determined bycytofluorometry), the CD3⁺ cell population was further enriched by cellsorting and expanding the cells by culture. After repeating the cellsorting twice, a CL3 population was obtained which stably expressed CD3.Fluorescent staining of the population with OKT3 and WT31 (anti-TcRαβ)indicated that essentially 100% of the cells expressed CD3 and themajority of the cells expressed TcRαβ. The CD3⁺ cell population and CD3⁻population were cultured and culture filtrates were assessed for thepresence of GIF by using the 12H5 cells. The GIF activity was detectedin culture filtrates of CD3⁺ cells, but not in the culture filtrates ofCD3⁻ population. The results indicated that the source of GIF is CD3⁺cells.

Since one of the unique properties of mouse GIF is that the monoclonalanti-lipomodulin (141B9) binds the lymphokine, it was decided todetermine whether human GIF from the CL3 cells would be absorbed with141 B9-coupled Sepharose. The CD3⁺, CL3 clone was cultured to yield 1liter of culture supernatant. After filtration through a YM100 membrane,the filtrates were concentrated to 5 ml, and fractionated on 1 ml 141-B9Sepharose. After recovering the effluent fraction, the immunosorbentcolumn volumes of 10 column volumes of DPBS, and then eluted with 5column volumes of glycine-HCl buffer, pH 3.0. After dialysis againstDPBS, distribution of GIF activity in the fractions was determined byusing the 12H5 cells. The results shown in Table II indicate thatessentially all GIF activity in the culture filtrate bound to 141-B9Sepharose and was recovered by elution at acid pH.

                  TABLE II                                                        ______________________________________                                        Human GIF From CL3 Clone Purified By Affinity                                 Chromatography On Anti-Lipomodulin Sepharose.sup.a                            Fraction from 141B9-       GIF Activity.sup.c                                 Sepharose.sup.b                                                                             Dilution     Effluent/Eluate                                    ______________________________________                                        Effluent      1:10         0/31 (-)                                           Washing       1:10         0/35 (-)                                           Eluate        1:10         42/0 (+)                                                         1:40         45/0 (+)                                                         1:80         39/0 (+)                                           Media Control --           0/34                                               ______________________________________                                         .sup.a Culture supernatants of the CL3 clone were filtered through YM100      membranes, and filtrates were concentrated 200fold. 5 ml of the               concentrated filtrate was fractionated on 1 ml 141B9Sepharose.                .sup.b After recovering the effluent fraction, the immunosorbent was          washed with 5 column volumes of DPBS, and then eluted with 5 column           volumes of glycine HCl buffer, pH 3.0.                                        .sup.c GIF activity was assessed by using the 12H5 cells by the same          procedures described in Table I. Numbers in the column represent percent      rosette inhibition by the effluent/eluate fractions from lentil lectin        Sepharose. The proportion of IgERFC in the absence of IgEBF was 22.9 ±     0.6 (SD) % in this assay. (+) indicated the presence of GIF.             

Previous experiments provided evidence that murine GIF is aphosphorylated derivative of a phospholipase inhibitory protein (Uede,et al., J. Immunol., 139: 898, 1983). Thus, GIF in the culture filtratesof CL3 clone was purified by using the 141B9-Sepharose. Culture filtrateof the three other clones, CL1, CL2, and CL7 were fractionated on the141B9-Sepharose in a similar manner. The acid eluates from theimmunosorbent were treated with alkaline phosphatase, and assessed forthe ability to inhibit the release of ³ H-oleic acid frombiosynthetically labeled E. coli by pancreatic phospholipase A₂ (Rothut,et al., Biochem. Biophys. Res. Commun., 117: 878, 1983). The resultsincluded in Table I indicate that the affinity-purified GIF from CL3 andCL7 exerted phospholipase inhibiting activity, while the same fractionfrom CL1 and CL2 failed to inhibit phospholipase A₂.

EXAMPLE 3 ANTIGEN-BINDING PROPERTIES OF GIF

Previous experiments have shown that antigen-activated T-cellspropagated with IL-2 in the presence of lipocortin constitutivelyreleased GIF that had no affinity for bee venom PLA₂, but cross-linkingof CD3 on the same cells resulted in the formation of GIF havingaffinity for the antigen-coupled Sepharose together with IgE-BF (Carini,et al., J. Immunol. Meth. 127: 221, 1990). In view of these findings, itwas decided to determine whether the CL3 clone produces antigen-bindingGIF and IgE-BF. The cells were treated with OKT3 at 0° C., and theantibody-treated cells (1.5×10⁶ cells/ml) were cultured in theanti-MGG-coated cells. As a control, untreated CL3 cells were culturedin the anti-MGG-coated wells. Culture supernatants were filtered throughYM100 membranes and concentrated seven-fold by ultra-filtration. Theconcentrated culture filtrates were absorbed overnight with 1 mlIgE-Sepharose, and unbound protein fraction and 2 ml of washings werecombined. The IgE-Sepharose was thoroughly washed, and eluted withglycine HCl buffer. The eluate fractions from IgE-Sepharose wereassessed for the presence of IgE-BF by using RPMI 8866 cells as thesource of FcεR⁺ cells.

                  TABLE III                                                       ______________________________________                                        Failure Of The GIF From The CL3 Clone                                         To Bind To Bee Venom PLA.sub.2                                                GIF Activity In PLA.sub.2 -Sepharose.sup.c                                               IgE-BF.sup.b                                                       Treatment.sup.a                                                                          (%)     Eluate     Washing                                                                              Eluate                                   ______________________________________                                        OKT 3      23      34/0(+)    21/0 (+)                                                                             0/24(-)                                  None        0      28/0(+)    22/13(±)                                                                          0/26(-)                                  ______________________________________                                         .sup.a Untreated or CD3treated cells were cultured in antiMGG-coated          wells.                                                                        .sup.b 30 ml culture supernatant were filtered through YM100, and             filtrates were concentrated to 4 ml. The samples were absorbed with 1.0 m     IgESepharose. Acid eluate fraction was adjusted to 4.0 ml and assessed fo     IgEBF by rosette inhibition. The proportion of IgEBF in the absence of        IgEBF was 37.7 ± 1.0%.                                                     .sup.c 1.0 ml of the effluent fraction from IgESepharose was fractionated     on PLA.sub.2Sepharose. The effluent, washing and acid eluate fractions        were adjusted to 1.3 ml, and were assessed for GIF activity by using the      12H5 cells. Numbers in the column represent percent rosette inhibition by     the effluent/eluate fractions from lentil lectin Sepharose. The proportio     of IgERFC in the absence of IgEBF was 21.7 ± 0.6 (SD) %.              

The results shown in Table III indicate that anti-CD3-treated cellsformed IgE-BF, while untreated cells failed to produce a detectableamount of IgE-BF. The effluent fraction from IgE-Sepharose wasconcentrated two-fold and 1 ml samples were fractionated on 0.25 ml PLA₂-Sepharose. The effluent fraction, washing, and eluate fraction wereadjusted to 1.5 ml, and the samples assessed for GIF activity. As shownin Table III, GIF from both unstimulated and anti-CD3 treated cellsfailed to bind to PLA₂ -Sepharose.

It was thought that the failure of the GIF from anti-CD3 treated CL3cells to bind PLA₂ might be related to the use of D-PLA₂ for theactivation of T-cells. In order to investigate this possibility, moreT-cell hybridomas from PBMC of a bee venom sensitive patient wereconstructed. The protocol for the construction of T-cell hybridomas wasexactly the same as that described above, except that PBMC werestimulated with 10 μg/ml CNBr-treated PLA₂ instead of D-PLA₂. As theresults of this experiment, 22 hybridoma clones were obtained. The GIFassay of culture filtrates of each clone indicated that 10 out of 22clones constitutively formed GIF (results not shown). SevenGIF-secreting clones were treated with OKT3 and the antibody-treatedcells were cultured in anti-MGG-coated dishes. Culture filtrates wereconcentrated four-fold and absorbed with IgE-Sepharose.

                  TABLE IV                                                        ______________________________________                                        Formation Of Antigen-Binding GIF By                                           Anti-CD3-Treated Hybridoma Cells.sup.a                                                              GIF Activity in                                                 IgE-BF.sup.b  PLA.sub.2 -Sepharose.sup.c                              Clone   (%)           Effluent       Eluate                                   ______________________________________                                        AC5     20            0/21 (-)   31/0 (+)                                     AF10    36            19/0 (+)   0/21 (-)                                     BA6      8            29/0 (+)   0/24 (-)                                     BE12    65            0/31 (-)   25/0 (+)                                     BF5     65            0/27 (-)   20/0 (+)                                     CB7     64            0/28 (-)   17/0 (+)                                     CE5     58            0/28 (-)   35/0 (+)                                     ______________________________________                                         .sup.a 1.2 × 10.sup.7 cells were treated with OKT 3. Cells were         resuspended in 8 ml culture medium and seeded in an antiMGG-coated flask.     Culture supernatant were concentrated fourfold and absorbed with              IgESepharose. Effluents from IgESepharose were then fractionated on           PLA.sub.2Sepharose and GIF activity in the effluent and eluate fraction       was determined.                                                               .sup.b Acid eluate fractions from IgESepharose were assessed for the          presence of IgEBF. The proportion of IgERFC in the absence of IgEBG was       26.3 ± 0.6 (SD) %.                                                         .sup.c GIF activity was determined by using the 12H5 cells. Numbers           represent the percent rosette inhibition by the effluent/eluate fractions     from lentil lectin Sepharose. Proportion of IgERFC in the absence of IgEB     was 26.0 ± 0.7 (SD) %. (+) indicates the presence of GIF.             

As shown in Table IV, acid eluate fraction from IgE-Sepharose of 6 outof 7 clones contained detachable amounts of IgE-BF. The effluentfractions from IgE-Sepharose were then fractionated on PLA₂ -Sepharose,and the effluent and eluate fractions from the immunosorbent wereassessed for GIF activity. The results shown in Table IV indicate thatthe majority of GIF from 5 out of 7 clones bound to PLA₂ -Sepharose andrecovered by elution at acid pH. In order to confirm that cross-linkingof CD3 is required for these clones to produce antigen-binding GIF, the5 clones were cultured in anti-MGG-coated cells without treatment withanti-CD3. As expected, culture supernatants did not contain IgE-BF, andGIF in the supernatant failed to bind to PLA₂ -Sepharose.

The present invention provides a technique to allow the development ofGIF-producing T-cell populations from PBMC of patients allergic to beevenom PLA₂, and to establish GIF-producing hybridomas from the T-cells.Representative hybridomas express CD3 determinants and TCRαβ, indicatingthat they are T-cell hybridomas. Furthermore, the TcR complex on thehybridomas appears to be functional. Both parent T-cells (Carini, etal., J. Immunol. Methods, 127: 221, 1990) and the majority of theGIF-producing hybridomas (Tables III, IV) produced IgE-BF uponcross-linking of CD3. Cross-linking of TcRαβ or CL3 and AC5 clones bythe monoclonal antibody WT31 and anti-MGG also resulted in the formationof IgE-BF (results not shown). Further testing of representative CD3⁺hybridomas showed that all of the CL3, BE12, AC5 and CB7 clonesexpressed both CD4 and CD8. Since BUC cells employed for construction ofthe hybridomas are CD4⁺ CD8⁻ (personal communication from Dr. J. Stobo),it is not clear whether the parent T-cells of the hybridomasco-expressed both CD4 and CD8.

The present experiments showed that some of the T-cell hybridomasproduced antigen(PLA₂)-binding GIF upon cross-linking of CD3 on thecells. This finding is in agreement with the fact that representativemurine GIF-forming hybridomas formed antigen-binding GIF uponstimulation with antigen-pulsed syngeneic macrophages or bycross-linking of CD3 on the cells (Iwata & Ishizaka, J. Immunol, 141:3270, 1988, Iwata, et al., J. Immunol., 143: 3917, 1989), and suggestedsimilarities between the antigen-binding GIFs from the two species. Inthe murine system, the antigen-binding GIF obtained from the hybridomassuppressed the in vivo antibody response in carrier (antigen)-specificmanner. It was also found that the antigen-binding GIF from thehybridomas were composed of antigen-binding polypeptide chain andnon-specific GIF (Jardieu and Ishizaka, in Immune Regulation byCharacterized Polypeptides, G. Goldstein, et al., ed., Alan R. Liss, NewYork, p.595, 1987), and that the antigen-binding chain shared a commonantigenic determinant 14-12 with those of the effector type suppressorT-cell factor (TseF) (Iwata, et al. ibid, 1989). Separate experimentshave shown that both the monoclonal anti-lipomodulin antibody 141-B9 andanti-I-J antibodies bound not only GIF, but also non-antigen bindingchain (I-J⁺ chain) of TseF and TsiF (Jardieu, et al., J. Immunol., 138:1494, 1986, Steele, et al., J. Immunol., 142: 2213, 1989). Thesefindings collectively suggest that the antigen-binding GIF is identicalto TseF. Parent T-cells of a representative murine Ts hybridoma 71B4were obtained by stimulation of OVA-primed spleen cells by homologousantigen, followed by propagation of the antigen-activated T-cells in thepresence of GIF. (Iwata & Ishizaka, J. Immunol., 141: 3270, 1988). Thesame strategy was employed to obtain the parent cells of the humanT-cell hybridomas in the present experiments. Indeed, both non-specificGIF and PLA₂ -binding GIF from the human hybridomas bound to141B9-Sepharose which previous studies had shown could also absorbmurine TsFs (Steele, et al., J. Immunol., 142: 2213, 1989). It could bethat PLA₂ -binding GIF from the human T-cell hybridomas represents humanantigen-specific TseF. However, it is still possible that theantigen-binding GIF may be a counterpart of murine TsiF. Recentexperiments in our laboratory have shown that the typical murine helperT-cell clone D10.G4.1 can produce antigen-binding GIF, if the cells wereprecultured in the presence of a phospholipase A₂ inhibitor, and thenstimulated with antigen(conalbumin)-pulsed antigen-presenting cells(Ohno, et al., Internat. Immunol, 2: 257, 1990). It was also found thatthis antigen-binding GIF bound to the monoclonal antibody 14-30, whichis specific for TsiF (Ferguson and Iverson, J. Immunol., 136: 2896,1986), rather than the monoclonal antibody 14-12. Green, et al., (J.Mol. Cell Immunol., 3: 95, 1987) also reported that D10.G4.1 cloneproduced antigen-binding TsF upon antigenic stimulation withUV-irradiated antigen-pulsed macrophages, and that this factor, togetherwith accessory molecules, induced the generation of the effector type,antigen-specific Ts. Since PBMC from allergic patients contain helperT-cells, it is still possible that the antigen-binding GIF from thehuman hybridomas represents TsiF rather than TseF.

Takeuchi, et al., (J. Immunol, 141: 3010, 1988) established Ts clonesfrom PBMC of KLH-primed individuals, who had received repeatedinjections of a large dose of homologous antigen. Modulin, et al.,(Nature, 322: 459, 1986) also established Ts clones from lesions oflepromatous leprosy patients. However, prior to the present invention,effector molecules mediating suppressor activity (TsF) from human Tscells have not been identified. Similarities between human GIF and mouseGIF suggest that the PLA₂ -binding GIF from human T-cell hybridomas mayrepresent TsF from human suppressor T-cells. The T-cell hybridomas,which produce antigen-binding GIF, will facilitate biochemicalcharacterization of the molecules. It has been repeatedly shown in themouse that Ts as well as TsF (antigen-binding GIF) suppressed the invivo IgE antibody response more effectively than the IgG antibodyresponse (Ishizaka, et al., J. Immunol., 114: 110, 1975). If theallergen-binding GIF from the human T-cell hybridomas actually representTsF, it is a reasonable expectation that the T-cell factor may suppressthe IgE antibody response of the donor of parent T-cells.

EXAMPLE 4 PREPARATION OF HYBRIDOMA CELL LINES PRODUCING CEDARPOLLEN-SPECIFIC GIF

Japanese cedar pollen is a major allergen in Japan and causes seasonalallergic rhinitis and conjuctivitis in a large percentage of thepopulation. In order to further test the general applicability of theteachings of the invention to other antigens, the methods for generatingantigen-specific GIF-producing T-cells and T-cell hybridomas (describedabove) were applied to peripheral blood mononuclear cells from patientsallergic to Japanese cedar allergen.

The major allergen in Japanese cedar (Sugi, Cryptomeria japonica) is a40 kDa glycoprotein designated cryj-1 (Yasueda, et al., J. Allergy andClin. Immunol., 71: 77, 1983). For these studies, the allergen wasisolated from extracts of cedar pollen by this method with slightmodifications. Briefly, pollen was defatted with ether, and extracted 3times with 0.125M ammonium bicarbonate. Carbohydrate in the extractswere removed by hexadecyltrimethyl ammonium bromide. Proteins in theextracts were precipitated with 80% saturated ammonium sulfate, and theprecipitate dissolved in 0.05M Tris-HCl buffer, pH 7.8. After extensivedialysis against the Tris-HCl buffer, the protein fraction was appliedto a DEAE cellulose column (DE-52, Whatman), and a flow-through fractionwas obtained. The fraction was concentrated, dialyzed against 0.01Macetate buffer, pH 5.0, and applied to a CM cellulose column (CM-52,Whatman), which was equilibrated with the buffer. The column was washedwith the buffer, and proteins retained in the column eluted with 0.1Mphosphate buffer containing 0.3M sodium chloride. Proteins in the eluatewere further fractioned by gel filtration through a Sephacryl S-200 HRcolumn to obtain a major protein fraction containing cryj-1. The majorprotein in the fraction was 42 kDa as determined by SDS-polyacrylamidegel electrophoresis, and N-terminal amino acid sequence of the proteinwas identical to that of cryj-1. The protein was conjugated to Affigel10 at 1.5 mg/ml gel.

A synthetic phospholipase A₂ inhibitor,2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, (ONO-RS-082, ONOPharmaceutical Co.) was used instead of recombinant human lipocortin I.Previous experiments had shown that ONO-RS-82 is a specific inhibitor ofphospholipase A₂ and facilitates the generation of GIF-producing cellsin mouse spleen cell cultures (Ohno, et al., International Immunology,1: 425, 1989). When spleen cells of ovalbumin-primed mice werestimulated with ovalbumin, and antigen-activated T-cells were propagatedwith IL-2 in the presence of either 2 μM ONO-RS-082, or 3 μg/mlrecombinant human lipocortin I, GIF-producing, antigen-specific T-cellswere generated. Antigen stimulated T-cells and construction of T-cellhybridomas were carried out essentially the same as described above,except that purified cryj-1 was used as antigen, and ONO-RS-082 wasemployed as a phospholipase A₂ inhibitor. Thus, mononuclear cells wereobtained from peripheral blood of patients allergic to Japanese cedarpollen, and suspended in RPMI 1640 medium containing 10% fetal calfserum (FCS). A suspension of the mononuclear cells (3×10⁶ cells/ml) werecultured for 3 days in the presence of 10 μg/ml cryj-1. Non-adherentcells were recovered, resuspended in RPMI medium containing 10% FCS,(3×10⁵ cells/ml), and cultured for 4 days in the presence of 60 units/mlhuman IL-2 and 2 μM ONO-RS-082. Cells propagated in this manner werethen recovered and fused with BUC cells to construct hybridomas.

Hybridomas were treated with the monoclonal anti-CD3 antibody SPB-T3b(Spits, et al., Hybridoma 2: 423, 1983), and the presence of CD3 on thecells were tested by immunofluorescence. Only CD3+ hybridomas weresubcloned by limiting dilution.

The CD3+ hybridoma clones were maintained in complete DME mediumcontaining 10% FCS, and culture supernatant of each clone was assessedfor the presence of GIF by using the 12H5 cells. Results obtained withhybridomas from one patient are shown in Table V. GIF activity wasdetected in culture supernatants of three hybridomas; 31E9, 31B7, and32B4. Supernatants of the other two hybridomas, 31H6 and 31H3, appear tohave weak GIF activity. Thus, the GIF-producing hybridomas were treatedwith anti-CD3 antibody followed by anti-mouse immunoglobulin, and thecells were cultured for 24 hr. Culture supernatants were thenfractionated on cryj-1 coupled immunosorbent. The presence of GIFactivity in the flow-through fraction and the acid-eluate fraction fromthe immunosorbent was assessed by using the 12H5 cells. The resultsincluded in Table V indicate that GIF from the 31E9 cells bound tocryj-1-Affigel and could be recovered by elution at acid pH, whereas GIFfrom the 31B7 cells failed to bind to the antigen-coupled immunosorbent.The results indicate that the 31E9 cells produce GIF having affinity forcryj-1, upon stimulation with anti-CD3.

                  TABLE V                                                         ______________________________________                                        PRODUCTION OF HUMAN CEDAR ALLERGEN-SPECIFIC                                   HYBRIDOMAS.sup.a                                                                                       GIF activity in cryj-1                               Hybridoma  % rosette inhibition.sup.b                                                                  Sepharose.sup.c                                      Clone      (effluent/eluate)                                                                           unbound   bound                                      ______________________________________                                        none       0/23          0/29      --                                         31H6       20/13(±)   5/20 (-)  12/10(±)                                31A11      0/25 (-)      ND        --                                         31E9       28/5(+)       0/22 (-)  20/0(+)                                    31H3       23/12(±)   0/34 (-)  38/16(±)                                31B7       32/5(+)       20/5(+)   4/24 (-)                                   31F7       0/26 (-)      ND        --                                         32B4       22/0(+)       22/14(±)                                                                             38/22(±)                                ______________________________________                                         .sup.a Hybridomas in this table were derived from two separate                experiments.                                                                  .sup.b Culture supernatants of unstimulated hybridomas were screened for      the presence of GIF. Aliquots of 12H5 cells were incubated with culture       supernatant of each hybridoma in the presence of mouse IgE. Culture           supernatants of the 12H5 cells were filtered through CF50A to remove IgE,     and filtrates were fractionated on lentil lectin Sepharose. IgEBF in the      effluent and eluate fractions was assessed by rosette inhibition. Numbers     in the column represent the percent rosette inhibition by the                 effleunt/eluate fractions from lentil lectin Sepharose. (+)(-) signs          indicate the presence or absence of GIF, respectively.                        .sup.c Representative hybridomas were treated with antiCD3 antibody and       culture supernatants were fractionated on cryj1 coupled Affigel. The          presence of GIF activity in the flowthrough (unbound) fraction, and acid      eluate (bound) fraction was determined by using 12H5 cells. Culture           filtrates of the 12H5 cells were fractionated on lentil lectin Sepharose.     Numbers represent percent rosette inhibition by the effluent/eluate           fractions from lentil lectin Sepharose. GIF from the 31E9 cells bound         toAffigel and was recovered by elution at acid pH, while GIF from the 31B     cells failed to be retained in the cryj1-Affigel column.                 

EXAMPLE 5 PREPARATION AND CHARACTERIZATION OF HYBRIDOMA CELL LINESPRODUCING MONOCLONAL ANTIBODIES SPECIFIC FOR HUMAN GIF

A. CONSTRUCTION AND SCREENING OF HYBRIDOMAS

Human GIF in culture supernatant of the T-cell hybridoma CL3 waspurified by using anti-lipomodulin (141-B9)-Sepharose. Theaffinity-purified GIF was mixed in complete Freund's adjuvant, andBALB/c mice were immunized by intraperitoneal injections of the antigen,given 3 times at 2 week intervals. Two weeks after the lastimmunization, spleen cells of the immunized mice were obtained, and1×10⁷ spleen cells were transferred into syngeneic BALB/c mice which hadbeen irradiated with 625R γ ray. The recipients were immunizedimmediately after cell transfer and 2 weeks later with purified GIFincluded in incomplete Freund's adjuvant. One week after the booster,their spleen cells were fused with HPRT-deficient B cell line SP2/0-14AG. The cells were cultured in HAT medium with BALB/c macrophagesas feeder layer. One hundred and two hybridoma clones obtained in theculture were selected for the formation of mouse immunoglobulin, andIg-forming hybridomas were selected for anti-GIF antibody production byELISA assay, followed by bioassay using the 12H5 cells.

In ELISA assay, Immulon I plates (Dynatech) were coated withaffinity-purified GIF. Control wells were filled with DPBS. Afterblocking the wells with 2% BSA, culture supernatants were applied toeach well, and the binding of mouse Ig to the wells was determined byusing alkaline-phosphatase-coupled anti-mouse Ig antibodies. As shown inTable VI, culture supernatants of 11 hybridoma clones gave a significantELISA signal.

                  TABLE VI                                                        ______________________________________                                        Selection of Anti-GIF-Producing Hybridomas.sup.a                              Hybridoma Ig         ELISA       GIF Activity.sup.c                           Clone       Isotype  Signal/Control.sup.b                                                                      Effluent/Eluate                              ______________________________________                                        none        --       0/0         29/1 (+)                                     334F        IgM      0.195/0.003 33/1 (+)                                     355C        IgM      0.388/0.012 28/0 (+)                                     338H        IgM      0.316/0.050 0/29 (-)                                     318H        IgM      0.149/0.046 0/31 (-)                                     388F        IgG      0.892/0.100 0/28 (-)                                     476B.sup.1  IgM.sup.2a                                                                             0.100/0.020 0/20 (-)                                     489G        IgM      0.174/0.00  7/15 (?)                                     481F        IgM      0.460/0.092 18/0 (+)                                     335C        IgM      0.203/0.073 0/27 (-)                                     419A        IgM      0.542/0.15  27/1 (+)                                     312F        IgM      0.533/0.029 14/8 (±)                                  Medium Control                                                                            --       0/0         0/31                                         ______________________________________                                         .sup.a Culture supernatants, which were positive in the ELISA assay, were     assessed for the ability to bind GIF from CL3 clone.                          .sup.b Binding of mouse Ig in culture supernatants of the hybridomas to       GIFcoated wells, as compared with nonspecific binding of Ig in the same       supernatants to BSAcoated wells. Optical density at 410 mμ.                .sup.c Mixtures of purified GIF with culture supernatants of hybridomas       were filtered through YM100 membranes, and the filtrates were assessed fo     GIF activity. The 12H5 cells were cultured with mouse IgE in the presence     of the filtrate. IgEBF formed by the cells was fractionated on lentil         lectin Sepharose and IgEBF in the effluent and eluate fractions from the      lectincoupled Sepharose were assessed by rosette inhibition. Numbers          represent the percent rosette inhibition by the effluenteluate fractions.     GIF switched the nature of IgEBG formed by the cells (top column vs.          bottom column). (+) indicates the presence of GIF.                       

The presence of anti-GIF in the culture supernatants of the 11 hybridomaclones was then determined by using the 12H5 cells (Iwata, et al., J.Immunol., 140: 2534, 1988). The globulin factor of culture supernatantfrom each clone was obtained by precipitation with 50% saturatedammonium sulfate. After dialysis against phosphate buffered saline, thefraction was adjusted to 1/5 volume of the original culture supernatant.Aliquots of an affinity-purified GIF prepared from CL3 clone using 141B9Sepharose. These aliquots were mixed with an equal volume of theglobulin fraction from each clone, and the mixtures were incubatedovernight at 4° C. The mixtures were then filtered through YM100membranes, and the presence of GIF in the filtrates was assessed. Then,aliquots of a suspension of the 12H5 cells were mixed with an equalvolume of the filtrate, and the cell suspensions were cultured for 24hours in the presence of 10 μg/ml mouse IgE. The culture supernatantswere filtered through CF50A membranes to remove IgE, and IgE bindingfactors in the filtrates were fractionated on lentil lectin Sepharose.The results of the experiments, included in Table VI, indicate that GIFwas removed by the culture supernatants of 338H, 318H, 388F₁, 476B, and335C clones, indicating that these hybridomas produce anti-GIF.

B. PURIFICATION OF HUMAN GIF WITH MONOCLONAL ANTI-GIF

Among the six hybridoma clones which produced monoclonal antibodies toGIF, only 388F₁ produced IgG antibody. This hybridoma was subcloned andcultured in high glucose Dulbecco's medium supplemented with 5% FCS.Culture supernatants were concentrated by ultra filtration and IgG inthe supernatants was recovered by using Protein A-Sepharose. Themonoclonal antibody was then coupled to Tresyl-activated Sepharose toprepare immunosorbent. In order to determine whether the monoclonalantibody could bind the same molecules as those bound toanti-lipomodulin (141B9) Sepharose, GIF in culture supernatant wasabsorbed with 141-B9-Sepharose, and was recovered by elution at acid pH.The affinity-purified GIF preparation was then fractionated withanti-GIF (388F₁)-coupled Sepharose. After the effluent fraction wasobtained, the immunosorbent column was washed with 10 column volumes ofDulbecco's phosphate buffered saline (DPBS), and then eluted withglycine HCl buffer, pH 3.0. A serial dilution of the effluent and eluatefractions were assessed for GIF activity by using the 12H5 cells. Theresults shown in Table VII indicate that GIF in the acid eluate fractionfrom 141B9-Sepharose bound to the anti-GIF (388F₁)-Sepharose and wasrecovered again by elution at acid pH. The results indicate that bothanti-lipomodulin and anti-GIF bind human GIF.

                  TABLE VII                                                       ______________________________________                                        Fractionation of Partially Purified                                           Human GIF on the Anti-GIF (388F.sub.1) Coupled Sepharose.sup.a                Fraction from              GIF Activity.sup.b                                 388F.sub.1 -Sepharose                                                                      Dilution      Effluent/Eluate                                    ______________________________________                                        Effluent     1:10          0/35 (-)                                                        1:20          0/29 (-)                                           Eluate       1:20          39/0 (+)                                                        1:40          26/0 (+)                                           Unfractionated                                                                             1:40          27/0 (+)                                           Media Control              0/27                                               ______________________________________                                         .sup.a GIF in culture supernatants of CL3 clone was purified by using the     antilipomodulin Sepharose. The affinity purified GIF (1.5 ml) was             fractionated on 0.75 ml of 388F.sub.1coupled Sepharose. After recovering      the effluent fraction, the column was washed with 10 column volumes of        DPBS, and then eluted with 3 column volumes of glycine HCl, pH 3.0.           .sup.b GIF activity was assessed by using the 12H5 cells by the same          procedures described in Table IV. Numbers in the column indicate the          percentage rosette inhibition by the effluent/eluate fractions from lenti     lectin Sepharose. (+) indicates the presence of GIF.                     

In order to determine if the anti-human GIF could bind mouse GIF, mouseGIF from Ts hybridoma, 231F₁ cells were purified by using141B9-Sepharose, and aliquots of the purified mouse GIF werefractionated on either 141B9-Sepharose or 388F₁ -Sepharose. After theeffluent fractions were obtained, immunosorbents were washed with 3column volumes of DPBS, and then eluted with 3 column volumes of glycineHCl buffer, pH 3.0. As expected, all GIF activity was absorbed to 141B9Sepharose, and recovered by elution at acid pH. Neither the effluent norwashing fraction contained GIF activity. When the same GIF preparationwas fractionated on 388F₁ -Sepharose, weak GIF activity was detected inthe effluent fraction. The majority of the activity was detected inwashings with DPBS, but the acid eluate fraction did not contain adetectable GIF activity. It appears that mouse GIF bind to anti-humanGIF with extremely low affinity, and disassociate from the immunosorbentby washing at neutral pH. These results indicate that the monoclonalantibody 388F₁ is specific for human GIF.

C. PURIFICATION OF HUMAN GIF BY ION EXCHANGE CHROMATOGRAPHY

AC5 cells were subcloned by limiting dilution and CD3⁺ clones obtained.These cells were then adjusted to serum-free ABC medium. Expression ofCD3 on the subclones cultured in the medium was confirmed byfluorocytometry. Culture supernatants of CD3⁺ subclones wereconcentrated 10-30 fold, and GIF activity in serial dilutions of thepreparations was determined. Based on these results, subclone (AC5-23)was selected, since a 1:3 dilution of the 10-fold concentratedsupernatant of this subclone could switch the 12H5 cells from theformation of glycosylated IgE-BF to the formation of unglycosylatedIgE-BF.

Studies were done to determine whether human GIF could be purified byion-exchange column chromatography. Culture supernatant of the AC5subclone in ABC medium was concentrated 25-fold. A 10 ml aliquot of theconcentrated culture supernatant was adjusted to pH 8.0 with Tris,diluted 8-fold with distilled water, and then applied to aDEAE-Sepharose column. Proteins bound to the column were eluted with 10mM Tris buffers containing increasing concentrations of NaCl. Eachfraction was concentrated to 10 ml and assessed for GIF activity.

                  TABLE VIII                                                      ______________________________________                                        Distribution of GIF Activity in DEAE-Sepharose Fractions                              Tris HCl +  Protein                                                   ______________________________________                                        Fraction                                                                              NaCl (mM) .sup.a                                                                          Content (μg) .sup.b                                                                   GIF ACTIVITY.sup.c                             ______________________________________                                        1        20         65.5       21/0(+)                                        2        50         35.0       20/6 (+)                                       3        75         42.5       7/20 (-)                                       4       100         38.5       3/19 (-)                                       5       150         41.5       0/21 (-)                                       6       200         42.0       0/20 (-)                                       medium                         0/22 (-)                                       control                                                                       ______________________________________                                         .sup.a Concentrated culture supernatants of the AC5 cells were diluted        8fold with distilled water, and applied to DEAESepharose column. Fraction     1 represents passed through fraction combined with washing with 10 mM Tri     HCl pH 8.0 containing 20 mM NaCl. The column was eluted stepwise with 10      mM Tris hCl containing increasing concentrations of NaCl.                     .sup.b Total protein recovered after concentration of each fraction. Afte     elution with Tris buffer containing 200 mM NaCl, much protein retained in     the column.                                                                   .sup.c GIF activity was detected by using the 12H5 cells. Numbers             represent the percent rosette inhibition by the affluent/eluate fractions     from lentil Iectin Sepharose. The proportion of RFC in the absence of         IgEBF was 22.6 ± 0.7 (SD) %. (+)(-) indicate the presence or absence o     GIF.                                                                     

As shown in Table VIII, the GIF activity was detected in thepassed-through fraction and in the eluate with 50 mM NaCl, but not inthe other fractions.

Titration of a serial dilutions of the first two fractions indicatedthat the pass-through fraction had higher GIF activity than the 50 mMfraction.

Repeated experiments with a separate culture supernatant confirmed thatthe majority of GIF in culture supernatants could be recovered from aDEAE-Sepharose column, when culture supernatant of AC5 cells wereconcentrated 100-fold, diluted 3-fold with distilled water, and thenpassed-through the column. The passed-through fraction and washings with10 mM Tris buffer containing 50 mM NaCl were combined, and concentratedto the original volume of the sample applied to DEAE-Sepharose.Titration of GIF activity in serial dilutions of the concentratedculture supernatant and the passed-through (50 mM NaCl) fraction showedthat a 1:30 dilution of both samples could switch the 12H5 cells fromthe formation of glycosylated IgE-BF to the formation of unglycosylatedIgE-BF. It was also found that 75 to 80% of protein in the culturesupernatant could be removed by passing through the DEAE-Sepharose.

In order to estimate the molecular mass of GIF, 0.5 ml of theconcentrated passed-through fraction from the DEAE-Sepharose was appliedto a Superose-12 column and proteins were eluted at a flow rate of 1ml/min. In this experiment, 5 ml fractions were collected, and eachfraction was assessed for GIF activity by using the 12H5 cells. GIFactivity was detected in fraction 9, which was recovered between 70 and75 min. Since Fractions 6 and 8 may also have a weak activity, GIFactivity in the serial dilutions of fractions 6, 8, 9 was assessed. TheGIF was detected in a 1:10 dilution of fraction 9, but not in a 1:2dilution of the other fractions. The results suggested that themolecular mass of the major species of human GIF is in the range of 11KDa to 18 KDa. For better estimation of the size of GIF molecules, gelfiltration on a Superose-12 was repeated in the same design, except that1 ml fraction or 2.5 ml fractions were collected. Three separateexperiments indicated that the majority of GIF was recovered between 68and 72 min. It appears that the molecular mass of GIF is 12-18 KDa, asestimated by gel filtration.

Studies were also done identifying GIF by SDS PAGE. Two liter culturesupernatant of the hybridoma in ABC medium were concentrated 100-fold,and fractionated on a DEAE-Sepharose column. Based on the experimentsdescribed above, the concentrated supernatant was diluted 3-fold withdeionized water and passed through the DEAE-Sepharose. Thepassed-through fraction was concentrated, pre-absorbed with humanIgG-coupled Sepharose, and GIF in the fraction was purified by affinitychromatography on the 388F1-coupled Affigel. In some experiments, theacid eluate fraction from the immunosorbent was adjusted to pH 8.0, andaffinity-purification with 388F1-Affigel was repeated. Analysis of theaffinity purified GIF preparation by SDS PAGE was performed underreduced and non-reduced conditions. The major band in theaffinity-purified material has the molecular mass of 14 KDa underreduced conditions and 15 KDa under non-reduced conditions. In addition,a 67 KDa band was frequently observed. A portion of theaffinity-purified preparation was dialyzed against DPBS and the GIFactivity in the preparation was titrated. Assuming 100% recovery of GIFduring dialysis and lyophilization, the sample applied to SDS-PAGEshould have a GIF titer of 1:250.

Experiments were carried out to determine the relationship between the14 KDa protein and GIF. The GIF in 2 liter culture supernatant of AC5clone was purified by DEAE-Sepharose chromatography followed byaffinity-purification using 388F1-Affigel. Acid eluates from theimmunosorbent was adjusted to pH 8.0, concentrated to 1 ml byultrafiltration and fractionated on a Superose 12 column. Every 2.5 mleluate fractions were assessed for activity by using the 12H5 cells. Inthis experiment, the majority of GIF activity was detected in thefraction eluated between 67.5 and 70 minutes. The presence of GIF in thefraction was confirmed by ELISA using biotin-coupled mAb 141-B9.Although the ELISA signal was weak, only the GIF-containing fractiongave ELISA signal. One ml of the GIF-containing fraction was lyophilizedand analyzed by SDS PAGE. The results confirmed that the 14 KDa peptideis present in the GIF-containing fraction.

EXAMPLE 6

PURIFICATION OF MURINE GIF AND AMINO ACID SEQUENCING

Murine GIF was purified from culture supernatant of GIF-producing murineT cell hybridoma 231F₁ cells using anti-lipomodulin monoclonal antibody141B9 (Iwata, et al., J.Immunol., 132: 1286, 1984). The monoclonalantibody 141B9 was purified from ascitic fluid of BALB/c mice injectedwith the hybridoma 141B9 by using a FAST-γ column. Approximately 10 mgof IgG₁ in the preparation were coupled to 1 ml of Affigel 10 beads. Inorder to obtain GIF, the 231F₁ cells were cultured in high glucoseDulbecco's modified Eagle's medium, supplemented with 10% Nu-serum(Collaborative Research). After the number of 231F₁ cells in the culturereached 1-2×10⁶ /ml, the cells were recovered, resuspended in serum-freeDMEM at the concentration of 1.5×10⁶ cells/ml, and cultured for 48 hr.Culture supernatants of the cells were concentrated 1000 fold by ultrafiltration, and 10 ml of the concentrated culture supernatant were mixedwith 2 ml of 141B9-coupled Affigel for 6-12 hr at 4° C. Theimmunosorbent was washed extensively with 10 mM phosphate buffercontaining 50 mM NaCl, followed by the same buffer containing 500 mMNaCl. Proteins retained in the immunosorbent were then eluted with 0.1Msodium acetate buffer, pH 3.0. Affinity-purified GIF was mixed with a1/10th volume of 100% (wt/vol) trichloracetic acid. The mixture was keptat -20° C. for 15 min, and centrifuged at 15,000×g for 5 min to recoverthe precipitates. Proteins in the precipitates were electrophoresed in15% polyacrylamide/SDS gel under reducing conditions, and electroblottedto polyvinylidene difuroride (PVDF) membrane in 10 mM CAPS buffer, pH11.0. After visualizing protein bands by staining with CoomassieBrilliant Blue (CBB), a 14 kDa band was excised for determination ofamino acid sequence.

The PVDF-immobilized protein was reduced and S-carboxymethylated insitu, and then digested with 1 pmol Achromobacter protease I (Wako PureChemicals) in 90 mM Tris buffer (pH 9.0) containing 8% acetonitrile for20 hours at 30° C. Digested peptides were separated by reverse-phaseHPLC using a 5 μC8-300A column (Waters) equilibrated with 0.05%trifluoroacetic acid in water as mobile phase. Peptides were eluted by alinear gradient (2 to 50%) of 0.02% trifluoroacetic acid in2-propanol/acetonitrile (7:3). Major peptide peaks showing absorbance at214 nm were collected and amino acid sequence analysis of the peptideswas performed using a gas-phase sequencer (Applied Biosystems Model470A) with modified program for micro sequencing (Iwamatsu, et al., J.Biochem., 110: 51-158, 1991). The amino acid sequences of the isolatedpeptides are shown below.

    ______________________________________                                        PEPTIDES    SEOUENCE                                                          ______________________________________                                        AP-1        (K)-I-G-G-A-Q-N-R-N-Y-S-K                                         AP-23       (K)-L-L-C-G-L-L-S-D-R-L-H-I-S-P-D-R-V-Y-                                      I-N                                                               ______________________________________                                    

PVDF-retained peptide fragments after Achromobacter protease I digestionwere sub-digested with 2 pmol of endoproteinase Asp-N (BoehringerMannheim) in 100 mM ammonium bicarbonate (pH 7.8) containing 8%acetonitrile for 16 hours at 30° C. After the digestion, four majorpeptides were collected by HPLC and sequenced as described above. Theamino acid sequence of each peptide is as follows.

    ______________________________________                                        PEPTIDES      SEQUENCE                                                        ______________________________________                                        AN-4          D-M-N-A-A-N-V-G-X-N-G-S-T-F-A                                   AN-5          D-P-C-A-L-C-S-L-H-S-I-G-K                                       AN-7          D-R-L-H-I-S-P-D-R-V-Y-I-N-Y-Y                                   ______________________________________                                    

X in AN-4 was not detected.

Peptides retained on the membrane after endoproteinase Asp-N digestionwere further sub-digested with 1 pmol trypsin-TPCK (WorthingtonBiochemical) in 100 mM ammonium bicarbonate (pH 7.8) containing 8%acetonitrile for 20 hr at 30° C. One major peptide (T-1) was collectedand sequenced.

    ______________________________________                                        PEPTIDES         SEQUENCE                                                     ______________________________________                                        T-1              P-M-F-I-V-N-T-N-V-P-R                                        ______________________________________                                    

The N-terminal amino acid sequence was directly sequenced by injecting asmall piece of PVDF-immobilized protein sequencer (Shimazu PSQ-1).

    ______________________________________                                        N-terminal    (M)-P-M-F-I-V-N-T-N-V-P-R-A-S-V                                 ______________________________________                                    

Approximately 85% of the analyzed peptides showed a deletion ofN-terminal methionine residue.

EXAMPLE 7 cDNA CLONING AND SEQUENCING OF MURINE GIF

Based on the N-terminal amino acid sequence and the sequence of anotherpeptide (AN-5) described above, oligo-nucleotides were synthesized.Attempts were made to amplify a partial cDNA by polymerase chainreaction, and to use the cDNA obtained to probe a murine cDNA library.The synthesized primers used in the PCR were: ##STR1## Cytoplasmic RNAwas isolated from GIF-producing murine T hybridoma 231F₁. Afterpurification of mRNA by using oligo(dT)-cellulose, single strand cDNAwas synthesized on the mRNA template by reverse transcription primed bydT₁₅ on 231F₁. RNA template. PCR was carried out in standard conditions.Briefly, the template DNA was denatured at 94° C. for 1 min, annealedwith the primers described above at 59° C. for 1 min, followed by anextension at 72° C. for 45 sec. A 0.2 Kb fragment amplified in the PCRwas ligated to pCR 1000 vector (Invitrogen, La Jolla, Calif.) forsubsequent cloning and DNA sequencing . After confirming the nucleotidesequence of the fragment, the insert was cut out with EcoRI digestion toscreen the cDNA library of murine T cell hybridoma, 231F₁ cells, whichwas constructed by using Uni-ZAP cDNA synthesis kit (Stratagene, LaJolla, Calif.). EcoRI recognition site was attached to double strandedcDNA, which was then digested with XhoI, and cDNA was ligated intoUni-ZAP XR vector. The cDNA library was screened by hybridization withthe 0.2 Kb DNA described above. Seven clones were isolated afterscreening a half million independent clones. Restriction mapping of allof the 7 clones showed a single pattern.

The longest clone (0.65 Kb) was chosen for DNA sequencing by a standarddideoxy method. The nucleotide sequence and deduced amino acid sequenceof murine GIF is shown in FIG. 1. Underlines indicate location of theidentified peptides in the Edman degradation of purified murine GIF.Estimated size of GIF protein is 13 kDa, which correlates with that ofpurified GIF from the T hybridoma 231F₁ cells. The nucleotide sequenceflanking the first methionine codon favors the translation initiationrule. The length of this insert was 0.65 Kb. Northern blot analysis ofmurine T cell and tissue RNA showed the presence of a single species ofmRNA at 0.65-0.70 Kb, suggesting that the obtained cDNA was full length.It was also found that the murine GIF protein lacks a signal peptide,since no methionine residues were found in the 5' upstream of nucleotide82 (FIG. 1).

EXAMPLE 8 ISOLATION OF cDNA ENCODING HUMAN GIF

Human T cell hybridoma AC5 was stained with anti-CD3 antibody and aGIF-producing CD3+ subclone was employed as a source of mRNA.Fractionation of RNA on oligo (dT)-cellulose was repeated to isolatemRNA, which was then employed as a template to synthesize cDNA using aZAP-cDNA synthesis kit. After an EcoRI recognition site was attached,double stranded cDNA was digested with XhoI and size selected byfiltration through Sephacryl S-400 spin columns. The cDNA was thenligated into the Uni-ZAP XR vector to construct a recombinant phagelibrary. In the library, the proportion of phage containing an insertwas 88 to 96% of total phage. The cDNA library was screened byhybridization with a fragment of cDNA encoding murine GIF. E. coli XL1were cultured with phagemid containing murine GIF-cDNA and DNA in thebacteria was extracted. Plasmid was purified by centrifugation anddigested with BamHI and XhoI. After electrophoresis on agarose, a 500 bpband was extracted, and purified by using Gene clean II kit (BIO 101).This cDNA was labeled with α-³² P-ATP using Prime-It gold kit fromStratagene.

E. coli PLK-F were cultured with the library which contained 5×10⁴ pfu,and phage were transferred to nylon membranes (Duralose, Stratagene)which had been coated with E. coli. The membranes were placed on an LBbottom plate and kept at 37° C. overnight. After treatment with 0.5MNaOH, neutralization with Tris and washing with 2×SSC, the membraneswere dried at 80° C. to fix DNA on the membranes.

For screening of the cDNA library, the membranes were pretreated withsonicated salmon sperm DNA, and then incubated overnight at 60° C. with³² P-labeled mouse GIF cDNA, which had been heated for 5 min at 100° C.The membranes were washed twice each with 6×SSC containing 0.1% SDS and0.1×SSC plus 0.1% SDS and exposed to radioautographic film. Phage wasextracted from positive clones and screening was repeated twice more toisolate positive clones. Among 200,000 phage which were screened, 27positive clones were isolated. In order to confirm that the positivephage clones actually contained cDNA homologous to mouse GIF cDNA,phagemid DNAs were obtained form each positive phage clone,electrophoresed in 1% agarose gel, blotted in Zeta-probe membrane andthen hybridized with ³² P-labeled mouse GIF cDNA.

In order to determine the nucleotide sequence of human GIF cDNA,phagemid from each phage clone was digested with EcoRI and XhoI, andelectrophoresed to obtain the insert. Among 27 clones, several cloneshaving a 0.5 Kb insert were sequenced in the dideoxy-method. The insertwas digested with SacI, PstI and SmaI and fragments were subcloned inpUC19 or pBluescript SK-vectors. Plasmid DNA was purified by thealkaline-SDS method and clones were sequenced using sequence Ver 2(USB). The entire nucleotide sequence of full length cDNA (PNY 106) isshown in FIG. 2. The sequence was homologous to the sequence of apurported human MIF cDNA (Weiser, et al., Proc.Natl.Acad.Sci.U.S.A. 86:7522-7526, 1989), except that the codon from nucleotides 390 to 392 isAAT (asparagine) in GIF cDNA, whereas the MIF cDNA has a codon of AGT(serine). Another difference was that 5' end noncoding region in pYN 106was 40 base longer than that of MIF.

An RNase protection assay was performed in several T hybridoma cells todetermine whether there was any redundancy in the structure of mRNAdetected by GIF cDNA. The results confirmed that there is only a singlespecies of mRNA corresponding to GIF.

EXAMPLE 9 EXPRESSION OF RECOMBINANT GIF IN E. COLI

A. Construction of bacterial expression systems

This example relates to expression of human and mouse GIF polypeptidesin E. coli. The human GIF cDNA inserted into BlueScript at EcoRI andXhoI sties was annealed with the oligonucleotide primers: ##STR2## Theseprimers were synthesized by the phosphoramidite method (McBride, et al.,Tetrahedron Lett., 24: 245-248, 1983). The 5'-end primer containedShine-Dalgano sequence for preferred bacterial expression (Scherer, etal., Nucl. Acids. Res., 8: 3895-3950, 1980), and each primer containedAfIII and BamHI sites, respectively.

The human GIF cDNA was amplified using the polymerase chain reaction(PCR) (Mullis, et al., Method in Enzymol, 155: 335-350, 1987). Unlessotherwise noted, the denaturation step in each PCR cycle was set at 94°C. for 1 min, and elongation was at 72° C. for 2 min. The temperatureand duration of annealing was variable from reaction to reaction oftenrepresenting a compromise based on the estimated requirement of severaldifferent PCRs being carried out simultaneously.

Amplified cDNA fragment, isolated from agarose gel, was digested withAfIII and BamHI, and ligation inserted into a pST811 vector carrying aTrp promoter and a TrpA terminator (FIG. 3, Japanese patent, Kokaikoho63-269983) at the unique AfIII and BamHI sites. This new plasmid, calledpTMK-hGIF (FIG. 4) was transformed into competent RR1 E. Coli hostcells. Selection of plasmid-containing cells was on the basis of theampicillin resistance (marker gene carried on the pST811 vector). TheDNA sequence of the synthetic oligonucleotides and the entire human GIFgene was confirmed by DNA sequencing of plasmid DNA.

The bacterial expression system of mouse GIF was constructed from themouse GIF cDNA inserted into BlueScript by the same procedures as humanGIF. The primers to generate AfIII and BamHI sites at both ends of mouseGIF cDNA using PCR were: ##STR3## The plasmid containing mouse GIF cDNAin pST811 vector was designated pTMK-mGIF.

Alternatively, the human or the mouse GIF coding sequence could berecovered from pTMK-hGIF or pTMK-mGIF by excision with AfIII and BamHI,and inserted into any desired bacterial vector, having a PL, lac, tac orOmpF promoter using procedures described in Molecular Cloning: ALaboratory Manual, Maniatis, et al., 1982. These exemplary bacterialvectors, as well as others known in the art, could be transformed intobacterial host cells and GIF expressed.

B. Culture of E. coli Producing GIF

RR1 E. coli carrying plasmid pTMK-hGIF or pTMK-mGIF were cultured in 20ml Luria broth containing 50 μg/ml of ampicillin, and grown overnight at37° C. The inoculum culture was aseptically transferred to 1 liter of M9broth which was composed of 0.8% glucose, 0.4% casamino acid, 10mg/liter thiamine and 50 mg/liter ampicillin, and cultured for 3 hrs at37° C. At the end of this initial incubation, 40 mg of indoleacrylicacid was added and the culture incubated for an additional 5 hours at37° C.

EXAMPLE 10 PURIFICATION OF RECOMBINANT GIF PRODUCTS EXPRESSED IN E. Coli

About 5 g wet weight of harvested cells were suspended in water to avolume of 30 ml and broken by French-Press (8000 psi repeated 4 times).Supernatants and broken cell pellets were separated by centrifugation at15000×g for 10 min at 4° C. The cell pellet was washed twice with water.By the use of SDS-polyacrylamide gel electrophoresis, it was evidentthat most of the pellet was human GIF protein. The supernatant alsocontained soluble human GIF protein. The ratio of soluble to insolubleGIF was about 1:3.

A. Purification of Soluble GIF

Soluble GIF fraction was frozen overnight at -80° C. and slowly thawedat room temperature. Insoluble material was removed by centrifugation at15000×g for 15 min. In this step, most of the bacterial contaminantscould be removed. The supernatant was adjusted to pH 6.0 by adding 50 mMsodium acetate buffer (pH 5.0) and applied to a CM-Sepharose Fast Flow(Pharmacia) column (5×18 cm) equilibrated with 20 mM sodium acetatebuffer (pH 6.0) at 4° C. The column was washed with 20 mM sodium acetatebuffer (pH 6.0) at a flow rate of 2 ml/min and proteins eluted by anNaCl step gradient. GIF was eluted with 0.5 NaCl in the 20 mM sodiumacetate buffer (pH 6.0). The purity of human GIF was estimated bySDS-polyacrylamide gel electrophoresis and determined to be more than95% pure.

B. Purification and refolding of insoluble GIF

The pellet fraction containing insoluble human GIF was suspended in 10ml of 0.2M Tris-HCl buffer (pH 8.0) containing 6M guanidine HCl and 25mM EDTA and incubated at room temperature for 3 hrs by gentle mixing tosolubilize human GIF. Remaining insoluble material was removed bycentrifugation for 15 min at 15000 g.

The soluble GIF fraction was applied to a Sephacryl S-200 Super Fine(Pharmacia) column (5×100 cm) equilibrated with 6M guanidine-HCl, 25 mMEDTA and 0.2M Tris buffer (pH 8.0) and eluted at a flow rate of 2 ml/minat room temperature. After the void volume had eluted, 10 ml fractionswere collected and analyzed for GIF by SDS-polyacrylamide gelelectrophoresis by Western blot staining. About 120 ml of GIF positivefractions were concentrated to 5 ml using a YM5 Milliporeultrafiltration membrane.

For refolding of the solubilized GIF, the sample was added slowly to 2liters of 20 mM Tris buffer (pH 8.0) with gentle stirring at roomtemperature. After 24 hours, the mixture was concentrated 10 fold usinga YM5 membrane.

For removal of remaining E. coli contaminants, a sample was applied to aTSK DEAE-5PWD (Toyo Soda) column (7.5×75 mm) equilibrated with 20 mMTris buffer (pH 8.0). After sample application, the column was washedwith the same buffer and GIF eluted with a gradient of 0 to 0.1M NaCl incolumn buffer at flow rate of 0.5 ml/min at room temperature.GIF-containing fractions, as determined by Western blot, wereconcentrated using a YM5 membrane. The purity of human GIF was estimatedby SDS-polyacrylamide gel electrophoresis and was determined to be morethan 95% pure. Recombinant mouse GIF was purified using the sameprocedures described above.

EXAMPLE 11 AMINO ACID SEQUENCING AND ANALYSIS OF RECOMBINANT GIF

A. Amino Acid Sequencing of Recombinant Human GIF

The purified recombinant human GIF obtained from a DEAE column wassubjected to SDS-polyacrylamide gel electrophoresis and blotted onto aPVDF membrane. The membrane was stained with Ponceau S and the GIF bandwas excised from the membrane. The N-terminal amino acid sequence wasdetermined by using a Shimazu PSQ-1 protein sequencer. About 60% ofrecombinant human GIF had the following 10 amino acids at theN-terminus:

    .sup.1 Met-.sup.2 Pro-.sup.3 Met-.sup.4 Phe-.sup.5 Ile-.sup.6 Val-.sup.7 Asn-.sup.8 Thr-.sup.9 Asn-.sup.10 Val-                    SEQ ID NO:7

This sequence was identical to the sequence deduced from the cDNAsequence of human GIF. About 40% of GIF lacked an N-terminal ¹ Metresidue.

B. Amino Acid Analysis of Recombinant Murine GIF

The purified recombinant mouse GIF obtained from a DEAE column washydrolyzed in twice-distilled 5.7M HCl containing 0.2% phenol for 24hours at 110° C. in an evacuated tube. This hydrolyzed sample wassuspended in 0.02M HCl and the amino acid composition determined(HITACHI 835S amino acid analyzer). In analyzing these results (TableIX), it was generally recognized that lower numbers of Cys, Thr, Met andTrp residues were obtained than was deduced from cDNA sequence due todegradation which occurs during acid hydrolysis. The lower value for Hisresidues could be due to insufficient separation from NH₃. Inclusion ofa known amount of an internal standard such as Leu in the amino acidcomposition analyses allowed quantitation of protein in the sample.Extinction coefficients of recombinant mouse GIF at 280 nm was 1.89.

                  TABLE IX                                                        ______________________________________                                        QUANTITATIVE AMINO ACID COMPOSITION                                           OF E. Coli DERIVED mGIF                                                                    AMINO ACID                                                                    COMPOSITION                                                                   MOLES                                                                         PER MOLE                                                         MOLECULE     OF PROTEIN  PREDICTED RESIDUES                                   AMINO ACID                                                                             RUN1    RUN2    RUN3  PER MOLECULE                                   ______________________________________                                        ASP + ASN                                                                              10.19   10.12   10.31 14                                             GLU + GLN                                                                              6.98    6.97    7.93  8                                              CYS      0.96    0.59    1.08  3                                              SER      9.11    9.03    9.12  9                                              GLY      8.98    8.97    8.78  9                                              HIS      0.000   1.70    1.86  3                                              ARG      5.07    4.81    4.84  4                                              THR      3.85    3.78    3.82  6                                              ALA      10.37   10.43   10.30 10                                             PRO      8.42    8.17    8.19  7                                              TYR      4.63    4.60    4.55  5                                              VAL      7.47    7.49    7.57  8                                              MET      3.13    3.03    3.05  4                                              ILE      6.14    6.09    6.10  6                                              LEU      11.00   11.00   11.00 11                                             PHE      4.28    4.02    4.35  4                                              TRP      0.00    0.00    0.00  1                                              LYS      2.92    2.95    2.87  3                                              ______________________________________                                    

EXAMPLE 12 PRODUCTION OF POLYCLONAL ANTIBODY AGAINST GIF

Three rabbits were injected subcutaneously with 100 μg of the same GIFsample in Freund's incomplete adjuvant every 2 to 3 weeks. After 114days, the rabbit serum was collected and the IgG purified by protein Aaffinity column chromatography (Prosep-A. BioProcessing). About 150 mgof IgG was obtained from 25 ml of serum. This antibody recognized mouseGIF and human GIF, and could be used for Western blotting andpurification of GIF.

EXAMPLE 13 EXPRESSION OF RECOMBINANT GIF IN MAMMALIAN CELLS

A. Construction of Mammalian Cell Expression Systems for DirectExpression

This example relates to expression of human GIF polypeptide in mammaliancells. The human GIF cDNA inserted into BlueScript at EcoRI and XhoIsites was annealed with the oligonucleotides primers: ##STR4## Eachprimer contained a BgIII or KpnI site. The human GIF cDNA was amplifiedusing PCR, isolated from agarose gel, and digested with BgIII and KpnI.This fragment was inserted by ligation into a modified SRα vector(Takabe, et al., Mol. Cell. Biol., 8: 466-472, 1988) having a BgIII sitefollowing a PstI site. The plasmid, called SRα-hGIF (FIG. 5) wastransformed into competent DH5 E. coli cells. Plasmid-containing cellswere selected on the basis of an Amp^(r) gene carried on the SRα vector.Plasmid DNA was isolated from cultured cells and the nucleotide sequenceof the entire human GIF gene was confirmed by DNA sequencing.

B. Construction of Mammalian Expression Systems Having an AdditionalSignal Sequence

Since GIF did not appear to have a signal sequence based on analysis ofthe DNA structure, another expression system was constructed tointroduce a signal sequence to GIF, so that the GIF polypeptide would besecreted from the transfected cells through a constitutive secretorypathway. Many secretory proteins, including polypeptide hormones, growthfactors and plasma proteins are synthesized as precursors and undergopost-translation proteolytic processing which is frequently required fortheir secretion and expression of biological activity. For example,human calcitonin is synthesized by endocrine C cells of the thyroid asthe large precursor molecule procalicitonon (Craig, et al., Nature, 295:345-347, 1982). Procalcitonin, which consists of N-terminal pro-region,calcitonin, and a C-terminal pro-region, undergoes proteolyticprocessing at the flanking dibasic sites to generate the calcitoninpeptide (Burns, et al., Mol. Endocrinol., 3: 140-147, 1989). Therefore,it was speculated that procalcitonin is cleaved by protein convertasesof neuroendocrine origin (Smeekens, et al., J. Biol. Chem. 265:2997-3000, 1990; Proc. Natl. Acad. of Sci., 88: 340-344, 1991). Inaddition, the N-terminal pro-region of human procalcitonin has anadditional Arg residue at the -4 position and has the sequence ofArg-Ser-Lys-Arg (amino acid residues 5-8 of SEQ ID NO:26) at the carboxyterminus which is a cleavage motif that can be recognized by theprocessing enzyme, furin (Fuller, et al., Science, 246: 482-486, 1989).

In the present example, human GIF cDNA was fused in-frame with the 3'end of the gene encoding the N-terminal pro-region of human calcitoninprecursor, and inserted into the SRα vector. Human furin cDNA was alsocloned and inserted into the SRα vector. Both vectors wereco-transfected to COS-1 cells (ATCC CRL 1650) which resulted insecretion of mature human GIF.

1. Cloning of cDNAs

The cDNA fragment encoding the signal peptide and N-terminal pro-regionof human pro-calcitonin (pro-CT) (Steerbergh, et al., FASEB Letter 207:94, 1986) was amplified by PCR using human calcitonin cDNA as template.mRNA was isolated from human thyroid carcinoma TT cells (ATCC CRL 1803),and reverse transcribed into cDNA which was used as a template for PCR.

Oligonucleotide primers having a PstI site, as shown below, weresynthesized and the human calcitonin precursor gene was amplified.##STR5## The amplified gene was cloned into PstI digested SRα vector.Human GIF cDNA inserted into BlueScript was annealed with theoligonucleotide primers shown below. ##STR6## The primers had a BgIIIsite and KpnI site, respectively. The amplified gene codes forArg-Ser-Lys-Arg sequence followed by the hGIF sequence. This gene wasthen inserted by ligation into BgIII and KpnI digested SRα which hadhuman calcitonin precursor gene as previously described. This newplasmid, designated SRα-hcGIF (FIG. 6) was transformed into DH5 E. colicells. Plasmid DNA was isolated from cultured cells and the DNA sequenceof entire human calcitonin precursor and the human GIF genes wereconfirmed by DNA sequencing.

Human furin cDNA was cloned by the same PCR amplification method.Poly(A)⁺ mRNA was isolated from the human bladder carcinoma cell lineHT1376 (ATCC, CRL 1472), reverse-transcribed into cDNA, and used as aPCR template. Six oligonucleotide primers (F1-F6, shown below) wereprepared based on the published furin cDNA sequence (Fuller, et al.,Science, 246: 482, 1989) using an ABI 394 DNA synthesizer (AppliedBiosystems, Inc. Calif.). Three cDNA fragments, covering codingsequences 1-951, 922-1604, and 1565-2385 bp of human furin DNA,respectively, were purified from the corresponding PCR products. ThecDNA fragment encoding the amino terminal protein sequence was annealedwith the adjoining cDNA fragment by using of a 27 bp overlap between thetwo fragments. The resulting cDNA mixture was re-amplified using primerscorresponding to the 5' end of the first fragment and the 3' end of thesecond fragment. The resulting 1.6 kb cDNA was ligated via the Bsp HIsite with the third cDNA fragment encoding the remaining carboxylterminal furin. The whole cDNA construct was subcloned into the TAcloning vector pCR1000 (Invitrogen, La Jolla, Calif.). Human furin cDNAsequences were determined using the Sequenase kit (United StatesBiochemical Corp., OH). Restriction site mapping and partial sequenceanalysis revealed that the cloned cDNA was identical to previouslyreported human furin. An Eco RI-Not I fragment containing thefull-length human furin was cloned into the mammalian expression vectorpEFneo, which was generated by inserting a neo-expression unit into amodified PEF-BOS (Mizushima, et al., Nucl. Acids Res., 18: 5322, 1990).

The six synthesized oligonucleotide primers used for cloning human furincDNA by PCR were: ##STR7## F1 and F2, were used to amplify the furincoding sequence of 1-951 and F3 and F4 were used to amplify 922-1604.Both PCR products were annealed by using a 27 bp overlap and theresulting cDNA mixture was re-amplified by using primers of F1 and F4.The derived cDNA of 1.6 Kb was ligated via the BspHI site with a F5 andF6 amplified cDNA fragment which encoded fragment 1565-2385 bp of thehuman furin gene. The entire furin cDNA was inserted into EcoRI digestedSRα vector. This new plasmid, designated SRα-hfurin, was transformedinto DH5 E. coli cells. Plasmid DNA was isolated from cultured cells andthe DNA sequence of the synthetic oligonucleotides and entire humanfurin gene was confirmed by DNA sequencing.

C. Expression of GIF in COS-1 Cells

The plasmids SRα-hGIF or SRα-hcGIF plus SRα-hfurin were transfected intoCOS-1 cells. Plasmid DNA was added to DMEM/F12 (1:1) medium containing10% Hanks' BSS, 2% FCS, 40 μg/ml DEAE dextran, 100 μM chlorquine at theconcentration of 1 μg/ml. This mixture was overlaid onto COS-1 cells inculture dishes and incubated (5 hours, 37° C., 5% CO₂). Afterincubation, cells were washed and cultured overnight (37° C. in DMEM/F12(1:1) medium with 10% FCS). After washing again, cells were cultured inserum free DMEM/F12 medium containing 20 μg/ml bovine insulin (Sigma),20 μg/ml human transferrin (Sigma), 40 mM monoethanolamine, 0.1 μMsodium selenite, and 1 mg/ml BSA for 1 week at 37° C. As a control, thevector without insert was transfected to COS-1 cells.

The amount of GIF in the culture supernatants was estimated by Westernblotting using anti-mouse GIF polyclonal antibodies. The supernatantsderived from SRα-hcGIF transfected COS-1 cells was shown to contain amature form of GIF. Furin expressed together with calcitonin-GIF cleavedthe calcitonin precursor sequence allowing the secretion of GIF. Theamount of GIF secreted from the COS-1 cells was comparable to the amountof calcitonin precursor-GIF secreted.

One skilled in the art can also construct other mammalian expressionvectors comparable to SRα-hGIF or SRα-hcGIF. The human GIF codingsequence could be recovered from SRα-hGIF or SRα-hcGIF by excision withBgIII and KpnI, and inserted by ligation into many vectors such as pCD(Okayama, et al., Mol. Cell. Biol., 2: 161,170, 1982), pCDM8 (Seed, etal., Proc. Natl. Acad. Sci. USA USA, 84: 3365-3369, 1987) and pDSVE(U.S. Ser. Nos. 025,344 and 152.045). The transformation of thesevectors into appropriate host cells, such as CHO, 3T3 and BHK cells, canresult in expression of GIF. It would be routine to select a preferredvector system, a GIF cDNA with or without signal sequence, and anappropriate host cell, for increasing secretion of GIF.

D. Construction of a unique fusion expression vector for the secretionof recombinant truncated peptide without co-transfection with furin cDNA

Furin is expressed in many tissues, and appears to be predominantlylocalized to the Golgi region (Bresnaham, P. A., et al., J.Cell.Biol.111: 2851, 1990; Mitsui, Y., et al., J. Biol. Chem., 266: 16954, 1991),suggesting that furin or a furin-like enzyme is involved in the cleavageof proproteins for the secretion of a mature protein through aconstitutive secretory pathway. The presence of a furin-like enzyme inCOS cells was predicted by Smeekens, et al., (Proc.Natl.Acad.Sci.,U.S.A., 89: 8822, 1992). Therefore, the furin-like enzyme in COS cellscan be utilized for processing of a recombinant fusion protein for thesecretion of a mature peptide, if a proper cleavage motif for the enzymeis used.

An Fc cDNA was utilized for test purposes to design an efficientproteolytic cleavage site. The Fc fragment of human IgG has no signalpeptide and the cDNA fragment encoding the Fc fragment does not carrythe translation initiation codon ATG, the protein cannot ordinarily beexpressed by transfection of the cDNA into mammalian cells. Pro-CT wasused for a carrier peptide for the Fc fragment, and amino acid sequencesof the carboxyl terminal end of the pro-CT were modified to create anappropriate cleavage motif which can be recognized by the putativefurin-like enzyme in COS-1 cells. Based on previous information oncleavage motifs for processing enzymes, four different amino acidsequences were introduced into pro-CT. The cDNA encoding pro-CT wasamplified by PCR with the human calcitonin cDNA as the template, usingone 5' end primer (CT1), and four different 3' end primers (CT2, CT3,CT4 and CT5) as listed in Table X. The primers CT2, CT3, CT4, and CT5were modified by introducing several basic residues in differentlocations in order to study the effect of such changes on the processingefficiency by the putative endoprotease of COS-1 cells.

                                      TABLE X                                     __________________________________________________________________________    LIST OF OLIGONUCLEOTIDE PRIMER                                                FOR PCR AMPLIFICATION OF HUMAN PRO-CT                                         __________________________________________________________________________                     Met                                                                              Gly                                                                              Phe                                                                              Gln                                                                              Lys                                                                              Phe                                           CT1(28 MER)                                                                          GAAT                                                                              TCT                                                                              GTC                                                                              ATG                                                                              GGC                                                                              TTC                                                                              CAA                                                                              AAG                                                                              TTC                                                                        |                                                                    ↓                                         CT2 (30 MER)  -6 -5 -4 -3 -2 -1 +1                                                   Leu Asp                                                                              Ser                                                                              Pro                                                                              Arg                                                                              Ser                                                                              Lys                                                                              Arg                                                                              Ser Arg                                              CTG GAC                                                                              AGC                                                                              CCC                                                                              AGA                                                                              TCC                                                                              AAG                                                                              AGA                                                                              TCT AGA                                              GAC CTG                                                                              TCG                                                                              GGG                                                                              TCA                                                                              AGG                                                                              TCT                                                                              TCT                                                                              AGA TCT                                                                       ←-----                                   CT3 (30 MER)                                                                         Leu Asp                                                                              Arg                                                                              Pro                                                                              Met                                                                              Ser                                                                              Lys                                                                              Arg                                                                              Ser Arg                                              CTG GAC                                                                              AGA                                                                              CCC                                                                              ATG                                                                              TCC                                                                              AAG                                                                              AGA                                                                              TCT AGA                                              GAC CTG                                                                              TCT                                                                              GGG                                                                              TAC                                                                              AGG                                                                              TTC                                                                              TCT                                                                              AGA TCT                                                                       ←-----                                   CT4 (30 MER)                                                                         Leu Asp                                                                              Arg                                                                              Pro                                                                              Arg                                                                              Ser                                                                              Lys                                                                              Arg                                                                              Ser Arg                                              CTG GAC                                                                              AGA                                                                              CCC                                                                              AGA                                                                              TCC                                                                              AAG                                                                              AGA                                                                              TCT AGA                                              GAC CTG                                                                              TCT                                                                              GGG                                                                              TCT                                                                              AGG                                                                              TTC                                                                              TCT                                                                              AGA TCT                                                                       ←-----                                   CT5 (30 MER)                                                                         Leu Asp                                                                              Ser                                                                              Pro                                                                              Met                                                                              Ser                                                                              Lys                                                                              Arg                                                                              Ser Arg                                              CTG GAC                                                                              AGC                                                                              CCC                                                                              ATG                                                                              TCC                                                                              AAG                                                                              AGA                                                                              TCT AGA                                              GAC CTG                                                                              TCG                                                                              GGG                                                                              TAC                                                                              AGG                                                                              TTC                                                                              TCT                                                                              AGA TCT                                                                       ←-----                                   __________________________________________________________________________     Arrow with dashed lines indicate the synthesized primer in the 5' and 3'      direction. CT1 encodes the amino terminus of proCT and the four other         primers encode the carboxyl terminus of proCT. Arrow with plain line          indicates the cleavage site by endoprotease. The nucleotide sequence          AGATCTAGA in CT2  CT5 is recognized by BgIII and XbaI.                   

In order to construct plasmids, an EcoRI restriction site was introducedinto CT1, and Bgl II plus Xba I restriction sites were introduced intofour other primers. After generating the four corresponding pro-CT cDNAfragments they were subcloned into the EcoRI and XbaI sites of theplasmid pBluescript II KS (+) (Stratagene). Since Not I became aneighbor restriction site after Xba I on this plasmid vector, the pro-CTcDNAs could be excised with EcoRI and Not I. Next, each of the fourdifferent pro-CT cDNAs with mutated 3'ends were inserted into themammalian expression vector pME18S, which carries the chimericretroviral promoter, SRα (Takabe, et al., Mol. Cell. Biol., 8: 466,1988). This new plasmid vector was designated pMEpro-CT (FIG. 7). Thisvector consists of i) a chimeric promoter SRα, fused by SV40 promoterplus the R region of long terminal repeat of the retrovirus, ii) pro-CT,and iii) SV40 early poly(A) addition signal. A multiple cloning site forBgl II, Xba I, and Not I was included, following the carboxyl terminalproteolytic cleavage site of pro-CT, for insertion of a foreign gene ofinterest. The nucleotide sequences (AGATCTAGA) recognized by Bgl II andXba I, encode Arg-Ser-Arg residues in the reading frame. Therefore,foreign cDNA can be introduced and fused with the pro-CT cDNA accordingto this frame.

The Fc cDNA of human IgG was obtained using PCR amplification methodwith two 28 nucleotide primers, G1 and G2, and with poly(A)⁺ mRNA from ahuman leukemia cell ine ARH-77 (ATCC, CRL1621) as the template. The 5'end primer G1 (5'-CTCTAGAGACAAAACTCACACATGC-3') and the 3' end primer G2(5'-GGCGGCCGCCGCACTCATTTACCCGGAG-3') contain an Xba I site and a Not Isite underlined respectively. The Fc cDNA thus obtained was subclonedinto the Xba I and Not I sites of the plasmid pBluescript II KS (+). ThecDNA fragment was sequenced using an ABI 270A sequencer (AppliedBiosystems, Inc., CA). The Fc cDNA encoded a sequence beginning at theAsp residue which is located after the first of three Cys residues inthe hinge region (Ellison, et al., Nucl. Acids Res., 10: 4071, 1982).The cDNA was inserted in-frame after the pro-CT region into pMEpro-CT.

1. Secretion and cleavage of the chimeric proteins

To test whether the fusion protein comprising of the pro-CT region andthe Fc fragment could be produced in a secreted form from theplasmid-transfected COS-1 cells, the chimeric plasmid containing cDNAencoding pro-CT with only dibasic Lys-Arg residues was used. Immunoblotanalysis of the culture supernatant of the transfected cells withanti-human IgG showed that the fusion protein was detected predominatelyat a molecular weight of approximately 66 kDa under non-reducingconditions and 37 kDa under reducing conditions. Similar immunoblotanalysis of the culture supernatant of the cell transfected with plasmidDNA of pMEpro-CT confirmed that the protein having reactivity withanti-IgG CT was not produced.

Based on the finding that the molecular size of the major protein in thenon-reduced form was approximately twice that found for the reducedform, it was hypothesized that the fusion protein must be synthesized asa dimer protein. Since no Cys residue exists in the pro-CT region, thedimer could arise from the disulfide bonds formed between the Cysresidues in the hinge region of the Fc fragment. Nevertheless, themolecular size of the protein (66 kDa as a dimer and 37 kDa as amonomer) indicated that the protein represents a fusion proteinconsisting of pro-CT and Fc fragment, but not the mature Fc fragment.

Processing efficiency was determined by transfecting the chimeric cDNAswith different cleavage motifs into COS-1 cells. Immunoblot analysisshowed that the Fc fragment, released by proper proteolytic cleavage,was not detectable when the chimeric plasmid contained pro-CT cDNAencoding the dibasic Lys-Arg motif at the cleavage site. All of thesecreted protein which reacted with anti-IgG CT antibodies was a fusionprotein of 37 kDa under reducing conditions. Similar results wereobtained when the fusion protein had an Arg residue at the P6 position,in addition to the Lys-Arg motif. When the fusion protein had an Argresidue at the P4 position, in addition to the Lys-Arg motif, the Fcfragment was detected in transfected COS-1 supernatant. However, themature Fc fragment represented only a small fraction of secretedproteins reactive to anti-IgG. In contrast, the fusion protein with theArg residues at both P4 and P6 positions at the carboxyl end of thepro-CT was processed most effectively to secrete a mature Fc fragment.When COS cells were transfected with the chimeric plasmid for theformation of the fusion protein with the cleavage motif ofArg-Pro-Arg-Ser-Lys-Arg, only a minute quantity of the fusion proteinand a large quantity of the fusion protein and a large quantity ofmature Fc fragment were detected in the supernatant. The resultsindicated that COS cells possess a processing enzyme which recognizedthe sequence of Arg-X-Arg-X-Lys-Arg.

2. Application of pMEpro-CT plasmid for the expression of bioactiverecombinant GIF

Since the experiment described above for the secretion of Fc fragmentindicated that the cleavage motif of a putative furin-like enzyme in COScells was Arg-X-Arg-X-Lys-Arg, this sequence was used for the formationof bioactive recombinant GIF. Pro-Ct cDNA was amplified by PCR using CT1and CT4 primers (see Table X), and the amplified cDNA was inserted intothe PME-pro-CT plasmid. The human GIF cDNA was introduced into theplasmid by methods described above for SRαhcGIF, and fused with thepro-CT cDNA. Transfection of the plasmid into COS cells resulted in thesecretion of the 13 kDa GIF.

EXAMPLE 14 PURIFICATION OF RECOMBINANT GIF PRODUCTS EXPRESSED INMAMMALIAN CELLS

For affinity purification, either the monoclonal anti-human GIF, 388F1(Thomas, et al., J. Immunol., 148: 729, 1992), or polyclonal rabbitantibodies against recombinant mouse GIF were coupled to Affigel 10(Biorad) or HiTrap NHS-activated column (Pharmacia) following theprocedures recommended by manufacturer. Monoclonal antibody (2-3 mgs) orγ globulin fraction of polyclonal antiserum (8-12 mgs) were coupled to 1ml gel.

Fractionation of culture supernatants on the immunosorbent was carriedout at 4° C. Culture supernatants of COS-1 cells were concentrated 10-20fold by ultrafiltration and 10-15 volumes of the concentratedsupernatant were passed through one volume of the immunosorbent columnovernight. When the volume of the concentrated material was limited, 2to 4 volumes of the supernatant were mixed overnight with one volume ofthe immunosorbent, and the suspension was packed into a column. Thecolumn was washed with 7-10 column volumes of PBS and proteins retainedin the column were eluted with 3 to 4 column volumes of 0.1M glycine HClbuffer, pH 3.0. After the pH was adjusted to pH 7-8 with Tris buffer,the samples were analyzed by SDS-PAGE and GIF was detected by eithersilver staining or western blot with polyclonal anti-recombinant GIF. Aportion of the wash fraction and acid eluated fraction were dialyzedagainst RPMI 1640 medium for bioassay. Purity of the 13 kDa protein inthe acid eluate fraction was higher than 90%.

Alternatively, mammalian cell-derived GIF was purified usingconventional column chromatography. In this example, 100 ml of COS-1supernatant was concentrated ten fold and applied to TSK G2000SW (ToyoSoda) column (21.5×600 mm) equilibrated with PBS. The column was run ata flow rate of 3 ml/min at room temperature. GIF was eluted in estimatedlow molecular weight fractions, as determined by Western blotting usingpolyclonal anti GIF antibodies. GIF-containing fractions were pooled andconcentrated by ultrafiltration and applied to a TSK DEAE-5PW (ToyoSoda) column equilibrated with 20 mM Tris HCl buffer, pH 8.0. The columnwas run at a flow rate of 0.5 mL/min at room temperature. Afterapplication, the column was washed with the same buffer, and GIF wasrecovered in this wash step. The differences in binding ability to DEAEbetween E. coli derived GIF and COS-1 derived GIF can be explained bythe existence of O-linked glycosylation or phosphorylation.

Further purification of GIF was carried out using Vydac Protein C4reverse phase column (The Separations Group) (4.6×150 mm). GIF fractions(10 ml) from the DEAE column was applied to C4 column, equilibrated with100 mM ammonium acetate buffer, and GIF purified with a gradient of 0 to90% ethanol in column buffer at a flow rate of 0.4 ml/min at roomtemperature. GIF was eluted in the fractions containing 50 to 60%ethanol, and was identified by SDS-polyacrylamide gel electrophoresisand Western blotting using polyclonal anti-GIF antibodies.

EXAMPLE 15 BIOLOGICAL ACTIVITIES OF RECOMBINANT GIF

A. Evaluation of GIF activity of recombinant GIF

The glycosylation inhibitory activity of recombinant human GIF wasevaluated by the ability of test samples to switch murine T cellhybridoma 12H5 cells from producing glycosylated IgE-binding factors(IgE-BF) to producing unglycosylated IgE-BF (Iwata and Ishizaka,J.Immunol., 141: 3270, 1988). In this assay, 12H5 cells were culturedfor 24 hr with 10 μg/ml mouse IgE in the presence or absence of a testsample. Culture supernatant was filtered through CF50A membranes toremove IgE. The filtrate was passed through a lentil lectin Sepharosecolumn and the column washed with 2 column volumes of DPBS. Proteinsretained on the column were eluted with 0.2M α methylmannoside. Theflow-through fraction combined with the wash and the eluate fractionwere each dialyzed for 2 days against DPBS, and the fractionsconcentrated. The presence of IgE-BF in the fractions was evaluated bythe ability of a fraction to inhibit rosette formation of FcεR+ B cellswith IgE-coated fixed ox erythrocytes by the procedures previouslydescribed (Yodoi, et al., J.Immunol., 124: 425, 1980). Mesenteric lymphnode cells of rats infected with the nematode, Nippostrongylusbrasiliensis were employed as a source of FcεR+ cells. When the 12H5cells were cultured with mouse IgE alone, essentially all IgE-BFproduced by the cells bound to lentil lectin Sepharose and was recoveredby elution with a methylmannoside. Thus, the ratio of the percentrosette inhibition between the effluent/eluate fraction was less than0.2. If a sufficient amount of GIF was added to the culture of 12H5cells together with mouse IgE, the majority of IgE-BF formed by thecells failed to be retained in the lentil lectin Sepharose, and wasrecovered in the effluent fraction. Thus, GIF in a test sample was takenas (+) if the ratio of the percent rosette inhibition between theeffluent/eluate fraction was 2.0 or higher.

Culture supernatants of COS-1 cells co-transfected with the plasmidSRα-hcGIF and SRα-furin have the ability to switch 12H5 cells fromproducing glycosylated IgE-BF to producing unglycosylated IgE-BF. Whenserial dilution of a 20-fold concentrated culture supernatant wasassessed for GIF activity by the method described above, the activitywas detected at the dilution of 1:100. A 4 ml aliquot of theconcentrated supernatant was fractionated on 2 ml 388F₁ (monoclonalanti-GIF)-coupled Sepharose; essentially all activity (>75%) wasrecovered in the acid eluate fraction, in which only the 13 kDa peptidewas detected. The protein was identified as GIF by Western blot.Titration of GIF bioactivity in flow-through and acid eluate fractionsindicated that the activity in the former fraction was less than 1/10 ofthat recovered in the eluate fraction. The concentration of this 13 kDaprotein in the maximal dilution of the eluate fraction for the detectionof GIF activity was approximately 10 ng/ml.

The acid eluate fraction from the 388F₁ Affigel was further fractionatedon Affigel-coupled with the y globulin fraction of polyclonalanti-recombinant mGIF. All GIF bioactivity and the 13 kDa peptide in thefraction bound to the immunosorbent and were recovered in the acideluate fraction. The concentration of the 13 kDa peptide in the fractionwas estimated by comparison with serial dilutions of E. coli-derivedrGIF as controls. The minimum concentration of the protein required forthe detection of GIF bioactivity was 5 ng/ml. The results collectivelyshowed that the active, recombinant hGIF bound to both monoclonalanti-human GIF and polyclonal antibodies against recombinant GIF (13 kDaprotein) and could be recovered by acid elution.

The same culture supernatant was fractionated on Sepharose coupled withthe monoclonal anti-lipomodulin 141-B9. Again, acid eluate fraction fromthe immunosorbent had GIF activity.

Since human GIF could be obtained by transfection of COS-1 cells withpME pro-CT-hGIF plasmid (Example 13, D2), culture supernatant of thetransfected COS cells was fractionated on 388F1 affigel, and the 13 kDapeptide recovered by acid elution was assessed for GIF biologicalactivity. 10-20 ng/ml of the 13 kDa peptide obtained by this method wassufficient for the detection of GIF activity. The results indicated thatthe procedure described in Example 13D is an effective method for theformation of highly bioactive GIF.

In order to determine whether the bioactivity of recombinant hGIF iscomparable to that of hybridoma-derived GIF, the GIF-producing human Tcell hybridoma, 31E9 was cultured in DMEM, and GIF in culturesupernatant was purified by using immunosorbent coupled with polyclonalanti-rGIF antibodies. The concentration of 13 kDa GIF in acid eluatefraction was estimated by Western blot, and the fraction was titratedfor GIF activity by using 12H5 cells. The results showed that 5-14 ng/mlof the 13 kDa peptide was sufficient to switch 12H5 cells to theproduction of unglycosylated IgE-BF. Thus, it appears that recombinantGIF is comparable to hybridoma-derived GIF with respect to the capacityto switch the glycosylation of IgE-BF. The experiments also show thathybridoma-derived GIF reacted with the antibodies to recombinant GIF.

Since previous experiments had shown that bioactivity of murinehybridoma-derived GIF increased by 3 to 10 fold upon treatment withalkaline phosphatase (Ohno, et al., Internat.Immunol., 1: 425, 1989), itwas decided to investigate this affect on the biologic activity ofrecombinant hGIF. Bovine serum albumin was added to affinity purifiedrGIF from 388F₁ -Affigel at the concentration of 2 mg/ml and dialyzedagainst 50 mM Tris HCl buffer (pH 8.2) containing 0.1M NaCl. One half ofthe preparation was mixed with sufficient insoluble alkaline phosphatase(Sigma) to give a concentration of the enzyme of 5 unit/ml, and thesuspension gently mixed for 2 hr at room temperature. After this time,the enzyme was removed by centrifugation, dialyzed against RPMI 1640medium, and GIF activity in the alkaline phosphatase-treated anduntreated samples assessed. These studies showed that untreatedrecombinant GIF switched the 12H5 cells to the production ofunglycosylated IgE-BF at a dilution of 1:30, but not at 1:60, while GIFactivity in the alkaline-phosphatase-treated sample was detectable atthe dilution of 1:200.

Recombinant GIF preparations obtained by transformation of E. coli orobtained by transfection of COS-1 cells with the plasmid SRαhGIF werecompared for bioactivity. Purified GIF derived from the soluble fractionof E. coli (see Example 10A) was further fractionated on 388F₁ -Affigel,or polyclonal anti-GIF-couped affigel, and recovered by elution at acidpH. All of the preparations gave a single band of 13 kDa in SDS-PAGE.The concentration of the 13 kDa GIF was estimated by silver staining andGIF bioactivity of the affinity-purified GIF was titrated by using the12H5 cells. The results showed that 100-200 ng/ml of GIF were requiredfor switching the cells from the production of glycosylated IgE-BF tothe production of unglycosylated IgE-BF. GIF from culture supernatantsof COS-1 cells transfected with the plasmid SRα-hGIF was also purifiedusing the 388F₁ -Affigel and assessed for GIF activity. The minimumconcentration of the 13 kDa GIF for switching the 12H5 cells wasapproximately 150 ng/ml (Table XI).

                  TABLE XI                                                        ______________________________________                                        BIOLOGICAL ACTIVITY OF AFFINITY-PURIFIED RECOMBINANT                          GIF                                                                                                                Minimum                                                                       concentra-                                                   Concentra-       tion of 13                               Recombinant                                                                            Immunosor- tion.sup.a       kDa protein.sup.c                        GIF      bent       μg/ml  GIF-titer.sup.b                                                                      ng/ml                                    ______________________________________                                        hcGIF    388F.sub.1 0.8       1:100  8                                                 388F1 fol- 0.2       1:40   5                                                 lowed by                                                                      poly anti-GIF                                                                 141B9      0.1       1:10   10                                       hGIF     388F.sub.1 1.5       1:10   150                                      E.coli-  388F.sub.1 2.0       1:20   100                                      derived GIF                                                                            unfraction-                                                                              10.0      1:10   1000                                              ated                                                                          poly anti-GIF                                                                            2.0       1:10   200                                      ______________________________________                                         .sup.a Concentration of the 13 kDa GIF protein in each preparation,           estimated by SDSPAGE.                                                         .sup.b GIF activity of the preparations were titrated by using the 12H5       cells.                                                                        .sup.c Minimum concentration of 13 kDa GIF required for switching the 12H     cells from the formatin of glycosylated IgEBF to the formation of             unglycosylated IgEBF.                                                    

B. Lack of macrophage migration inhibitory activity

Because of the homology between GIF and MIF in cDNA nucleotide sequences(Weiser, et al., supra, 1989), GIF was tested for MIF activity. HumanMIF inhibits migration of human monocytes and the macrophages of guineapigs and mice, therefore the activity of recombinant hGIF was determinedusing mouse macrophages. Three ml of sterile mineral oil was injectedintraperitoneally into normal BALB/c mice and peritoneal exudate cellswere recovered 3 days later. Mononuclear cells were recovered bycentrifugation through a FCS layer, and a cell pellet containingapproximately 5×10⁷ mononuclear cells was suspended in 50 μl ofprewarmed 0.35% agarose in DMEM containing 15% FCS. One μl droplets ofthe suspension were dispersed into the center of each well of aflat-bottomed 96-well microtiter plate, which was kept for 5 minutes onice. DMEM supplemented with 15% FCS was added to the wells, togetherwith a sample to be tested in a total volume of 100 μl. One test samplewas assessed in triplicate or quadruplicate. The diameter of eachdroplet was measured under an inverted microscope. After incubation for20-24 hours, the diameter of the outer area of the migrating cells wasmeasured, and the migration index was calculated. The percent inhibitionof migration was calculated by the following formula:

    Percent inhibition= 1-migration index of test sample\migration index of migration without sample!×100

The MIF activity of rhGIF was assessed using mouse γ-interferon as apositive control. Serial dilutions of recombinant hGIF obtained bycotransfection of COS-1 cells with SRα-hcGIF and SRα-hfurin, andaffinity purified by using 388F1-affigel as described above. Even thoughthe GIF titer of the preparation was 1:100, no significant inhibition ofmacrophage migration was detected at the final dilution of 1:8 or 1:4.In the same experiments, 1 unit/ml of IFNγ inhibited microphagemigration by 20 to 48%.

The results were confirmed by using human monocytes in agar droplets(Remold, H. G. and Menddis, A. D., Methods in Enzymology 116: 379,1985)using human MIF as a positive control. The recombinant hGIF which couldswitch the 12H5 cells for the formation of unglycosylated IgE-BF at adilution of 1:100 and contained approximately 0.8 μg/ml of the 13 kDaGIF peptide, failed to inhibit the migration of human monocytes at thefinal dilution of 1:5. The results indicated that rhGIF was differentfrom MIF in biological activity.

EXAMPLE 16 IN VITRO GENERATION OF ANTIGEN-SPECIFIC SUPPRESSOR T CELLS BYRECOMBINANT hcGIF

Previous experiments have shown that GIF from the rat-mouse T cellhybridoma 23A4 cells facilitated the generation of antigen-specificsuppressor T cells from antigen-primed spleen cells (Iwata, M. andIshizaka, K; J. Immunol. 141: 3270, 1988). When BDF₁ mice were immunizedwith alum-absorbed ovalbumin for IgE antibody response, their spleencells constitutively secrete glycosylation enhancing factor (GEF) andproduced both IgE potentiating factor (glycosylated IgE-BF) andantigen-binding GEF upon antigenic stimulation. If the antigen-primedspleen cells were stimulated by ovalbumin, and antigen-stimulated Tcells were propagated by IL-2 in the presence of GIF, the cells secretedGIF. Upon antigenic stimulation, these cells produced unglycosylatedIgE-BF and antigen-binding GIF, the latter of which suppressed theantibody response of syngeneic mice to DNP-OVA in carrier-specificmanner. In order to explore this phenomenon more fully, it was decidedto determine whether recombinant hcGIF may have the ability to generateGIF-producing T cells in vitro.

BDF₁ mice were primed with 1 μg ovalbumin (OVA) absorbed to aluminumhydroxide gel for IgE antibody response. Two weeks after immunization,spleen cells were obtained, and a suspension (5×10⁶ cells/ml) culturedfor 3 days with 10 μg/ml OVA to activate antigen-primed T cells. Analiquot of the antigen activated cells (2.5×10⁵ cells/ml) were thenpropagated using recombinant mouse IL-2 (50 units/ml) in the presence orabsence of rhcGIF (0.25 μg/ml). After a 4-day culture, the cells wererecovered, washed 3 times, and resuspended in fresh culture medium atthe concentration of 1.5×10⁶ cells/ml. Four ml of the cell suspensionrecovered from GIF (+) or GIF (-) cultures were incubated for 24 hourswith 8×10⁵ OVA-pulsed LB27.4 cells which have both Ia^(b) and Ia^(d)(American Type Culture Collection, Rockville, Md.). Culture supernatantsof antigen-stimulated T cells were absorbed with IgE-coupled Sepharose,and IgE-BF was recovered from the immunosorbent by acid elution. Theflow-through fraction from the IgE-Sepharose was then fractionated onOVA-coupled Sepharose, and OVA-binding factors were recovered by elutionwith glycine-HCl buffer, pH 3.0. Eluates from IgE-Sepharose (i.e.,IgE-BF) were fractionated on lentil lectin Sepharose to determine thenature of the factors. The results shown in Table XII indicate that themajority of IgE-BF from the T cells propagated by IL-2 alone bound tolentil lectin Sepharose and were recovered by elution witha-methylmannoside. In contrast, T cells propagated by IL-2 in thepresence of hcGIF formed unglycosylated IgE-BF, which failed to beretained on lentil lectin Sepharose. Association of GEF or GIF activitywith OVA-binding factors was also assessed. Upon antigenic stimulation,T cells propagated with IL-2 alone formed OVA-binding GEF, whereas Tcells propagated in the presence of recombinant GIF formed OVA-bindingGIF (Table XII). It has been shown that OVA-binding GIF from such Tcells suppressed the antibody response of BDF₁ mice in an antigen(carrier)-specific manner (Iwata, M. and Ishizaka, K; J. Immunol.,1988). Thus, the recombinant GIF facilitated the generation ofsuppressor T cells which produced antigen-specific suppressor T cellfactor.

                  TABLE XII                                                       ______________________________________                                        GENERATION OF GIF-PRODUCING CELLS                                             BY RECOMBINANT hcGIF AND IL-2                                                                          OVA-binding                                          Propagation of OVA-      factor.sup.c                                         specific T cells.sup.a                                                                      Nature of IgE-BF.sup.b                                                                       GEF    GIF                                       ______________________________________                                        IL-2          4/28           +      -                                         IL-2 + hcGIF  26/6           -      +                                         ______________________________________                                         .sup.a Antigenstimulated T cells were propagated with IL2 in the presence     or absence of recombinant hcGIF.                                              .sup.b T cells propagated with IL2 were stimulated with antigenpulsed         LB27.4 cells. IgEBF in the supernatants were fractionated on lentil lecti     Sepharose. Numbers represent percent rosette inhibition by the                effluent/eluate fractions from elntil lectin Sepharose, respectively.         .sup.c Acid eluate fraction from OVASepharose was assessed for GIF and GE     activities by using the 12H5 cells and 23A4 cells, respectively.         

EXAMPLE 17 IN VIVO ACTIVITY OF RECOMBINANT GIF

In vivo activity of recombinant GIF

Previous experiments have shown that repeated injections of aGIF-enriched fraction of culture filtrates of GIF-producing hybridomainto immunized mice beginning on the day of priming resulted insuppression of both IgE and IgG antibody responses (Akasaki, M., et al.,J. Immunol. 136: 3172, 1986). In order to determine whether recombinantGIF has the same in vivo effects, rhGIF expressed in E. coli (Example10A) and that expressed in COS-1 cells (Example 13A), were purified tohomogeneity by the methods described in Examples 10 and 14, and assessedfor their ability to suppress the IgE and IgG1 antibody responses. BDF₁mice were immunized by an i.p. injection of 0.2 μg DNP-OVA absorbed to 2mg alum. Recombinant GIF was injected i.p. on day 0, 2, 4, 6, 8, 10 and12, and anti-DNP antibodies in serum were measured by ELISA.

In the first experiment, E. coli-derived rGIF in PBS was administered ata dose of 18 μg/injection and control mice received PBS alone. Minimumconcentration of rGIF for switching the 12H5 cells from the formation ofglycosylated IgE-BF to the formation of unglycosylated IgE-BF wasapproximately 1 μg/ml. In both control and GlF-treated mice, IgEantibody titer reached maximum at 2 weeks and then declined, while IgG1anti-DNP antibody titer continued to increase for 4-5 weeks after theimmunization. Thus, IgE antibody titer at 2 weeks and IgG1 antibodytiter at 4 weeks were used for comparison between the GIF-treated anduntreated groups.

    ______________________________________                                        Effect of E. coli Derived rGIF                                                Sample   N          Anti-DNP IgE.sup.a                                                                         IgG1.sup.b                                   ______________________________________                                        control  6          6.7 ± 3.5 78.0                                         rGIF     3          1.5 ± 1.3 (p < 0.05)                                                                    4.5                                          ______________________________________                                         .sup.a 2 weeks after immunization (μg/ml)                                  .sup.b 4 weeks after immunization (μg/ml).                            

In the second experiment, COS-1 derived rhGIF, which was expressed byusing SRα-hcGIF vector, was administered i.p. at a dose of 2.5μg/injection. The minimum concentration of hGIF for switching the 12H5cells for the formation of unglycosylated IgE-BF was 0.2 μg/ml. Controlmice received PBS alone. The results are shown below.

    ______________________________________                                        Effect of Mammalian Cell Line Derived rGIF                                    Sample   N             anti-DNP IgE.sup.a (μg/ml)                          ______________________________________                                        control  8             2.6 ± 1.2                                           rGIF     5             0.70 ± 0.55 (p < 0.05)                              ______________________________________                                         .sup.a 2 weeks after immunization.                                       

None of the animals had adverse reactions to GIF. The exampleillustrates immunosuppressive effects of GIF.

EXAMPLE 18 PRODUCTION OF mAB TO ANTIGEN-SPECIFIC GLYCOSYLATIONINHIBITING FACTOR

A. MATERIALS AND METHODS

1. Antigens and antibodies: Lyophilized bee venom phospholipase A₂(PLA₂) was from Sigma Chemical Co., St. Louis, Mo. Crystalline ovalbumin(OVA) was from Nutritional Biochemical Corp., Cleveland, Ohio. Syntheticpeptide corresponding to amino acid residues 13-28, and thatcorresponding to amino acid residues 25-40 in PLA₂ molecules weresupplied by Dr. Howard Grey (Cytel Corp., La Jolla, Calif.). The peptidecorresponding to amino acids 19-35 in PLA₂ molecules was synthesized byKirin Pharmaceutical Laboratory, Maebashi, Japan. All of the syntheticpeptides were purified by HPLC, and their amino acid sequences wereconfirmed.

The monoclonal anti-human GIF antibody, 388F₁ (ATCC HB 10472), wasdescribed in Examples 5.

2. Cell lines: The human T cell hybridoma AC5 cells described in Example3 expresses both CD3 and TCRαβ, and constitutively secretes GIF. Thecells were cultured in high glucose DMEM supplemented with 10% FCS, 10%NCTC 135 medium (Gibco, Grand Island, N.Y.), 10 mM HEPES buffer, 0.2u/ml bovine insulin (Sigma), 50 μg/ml sodium pyruvate, 150 μg/mloxaloacetic acid and antibiotics.

B cell hybridomas that produce anti-human GIF were constructed fromspleen cells of BALB/c mice (Jackson Lab, Bar Harbor, Me.) that had beenimmunized with affinity-purified antigen-binding GIF (see below). Oneweek after the last booster immunization, the spleen cells were fusedwith hypoxanthine phosphoribotransferase-deficient Sp2/0 AG14 cells(Schulman, et al., Nature, 271: 269, 1978) by the procedures described(Iwata, et al., J. Immunol., 132: 1286, 1984). Subcloning of thehybridomas was carried out by Imiting dilution. The hybridomas weremaintained in complete DMEM described above.

3. Fractionation and purification of GIF: Nonspecific GIF in culturesupernatants of unstimulated AC5 cells in serum-free medium was enrichedby chromatography on a DEAE Sepharose column. Culture supernatants wereconcentrated 20 to 100 fold, diluted 3 fold with distilled water,adjusted to pH 8.0 with Tris, and then passed through a DEAE Sepharose(Pharmacia) column which had been equilibrated with 10 mM Tris HClbuffer, pH 8.0, containing 50 mM NaCl. Flow-through fraction and washingof the column with the buffer were combined and concentrated.Nonspecific GIF in the original culture supernatant wasaffinity-purified by using 388 F₁ -coupled Affigel. Concentrated culturesupernatant was recycled overnight through the immunosorbent. Afterwashing with 30 column volumes of DPBS, proteins bound to theimmunosorbent were recovered by elution with 0.1M glycine HCl buffer, pH3.0.

To produce antigen-binding GIF, AC5 cells were stimulated bycross-linking of CD3 (Thomas, et al., J.Immunol. 148: 729, 1992). Thecells (5×10⁶ ml) were treated with 5 μg/ml of monoclonal anti-CD3(SPV-T₃ b) for 30 minutes at 4° C., and the antibody-treated cells wereincubated for 30 minutes at 4° C. with 10 μg/ml anti-mouse Ig. Afterwashing, the cells were resuspended in ABC medium at 2×10⁶ cells/ml, andcultured for 24 hours. Culture supernatants were concentrated 10 to 15fold by ultrafiltration, and recycled overnight through a PLA₂ -coupledSepharose column. The column was washed with DPBS and proteins remainingin the column were recovered by elutin with 0.1M glycine HCl buffer, pH3.0.

Affinity purified GIF was fractionated by gel filtration through aSuperose 12 column (1.6×50 cm, Pharmacia) connected to HPLC (BeckmanSystem Gold, Fullteron, Calif.). Proteins were eluted from the columnwith PBS at a flow rate of 0.85 ml/min. The column was calibrated withBSA (m.w. 67,000), OVA (m.w. 43,000), soybean trypsin inhibitor (m.w.20,100) and cytochrome c. (m.w. 12,500). In some experiments,affinity-purified GIF preparation was reduced and alkylated. The GIFpreparation in 0.05M Tris HCl buffer containing 0.15M NaCl was incubatedfor 1 hour at room temperature with 10 mM dithiothreitol and thenalkylated with 30% molar excess of iodoacetamide. The reduced andalkylated sample was applied to the same Superose 12 column, andproteins were eluted with PBS.

4. ELISA assays: Each well of a Nunc F plate (Max Sorp, Nunc) was coatedwith 50 μl of serial dilutions of a GIF preparation overnight at 4° C.in duplicate or triplicate. Plates were washed five times with phosphatebuffered saline containing 0.05% Tween 20 (Sigma) between each of thefollowing steps except the step prior to addition of substrate. Theplates were blocked with 2% BSA in Tween/PBS for 1 hour at 37° C.Binding of mAb 388F₁ to GIF was detected with an amplification system.Fifty μl of PBS containing 150 ng/ml of biothinylated mAb 388F₁ wereadded to each well. After 2 hours of incubation at 37° C., followed bywashing, 50 μl of an appropriate dilution (1:6000) ofstreptavidin-coupled alkaline phosphatase (Zymed) was added to each welland the plate was incubated for 2 hours at 37° C. The plate was washedwith 0.05% Tween 20 in 0.05M Tris HCl buffer, pH 7.5, containing 0.15MNaCl, and an ELISA signal was developed by 30 min incubation with 50 μlof alkaline phosphatase substrate followed by amplifier solution(GIBCO/Bethesda Research Lab). Absorbances at 490 nm was determined inan ELISA reader MR 5000 (Dynatech).

ELISA was also set up with mAB against antigen-binding GIF. AfterMax-Sorp plates were coated with a GIF-preparation and blocked with BSA,50 μl of the mAb (200 ng/ml) in PBS was added to each well and the platewas incubated for 2 hours at 37° C. Depending on the isotype of the mAb,a 1:3000 dilution of horse radish peroxidase (HRP)-coupled goatanti-mouse IgM (Biorad) or anti-mouse IgG (Zymed) or a 1:2000 dilutionof HRP-coupled anti-mouse IgG+A+M (Zymed) was added to each well. ELISAsignal was developed by peroxidase substrate (Zymed), and determined byabsorption at 405 nm.

5. SDS-PAGE and immunoblotting: Affinity-purified GIF was dialyzedagainst 0.01% SDS and lyophilized. Samples were then analyzed by SDS gelelectrophoresis in 15% polyacrylamide slab gel by using the Laemelisystem (Laemeli, U.K., Nature, 227: 680, 1980). Protein bands weredetected by silver staining (Ochs, et al., Electrophoresis, 2: 304,1981). For immunoblotting, affinity-purified GIF was analyzed bySDS-PAGE under reducing conditions. Purified recombinant GIF from E.coliwas electrophoresed in parallel as a standard. Proteins in SDS-PAGE gelwere blotted to PVDF membrane (Immobilon-P, Millipore), and the membranewas blocked by incubating with Blocker Blotto (Pierce) overnight at 4°C. After washing with 0.05% Tween 20 in PBS, pH 7.5, membrane wastreated with 1 μg/ml of IgG fraction of rabbit anti-rGIF for 1 hour at37° C. Binding of rabbit IgG to protein bands was detected by usingHRP-coupled donkey anti-rabbit IgG and ECL Western blotting detectionreagents (Amersham), followed by autoradiography. The position of rGIFband on x-ray film was used as 14 kDa standard.

B. Preparation of monoclonal antibodies specific for antigen-binding GIF

AC5 cells were treated with anti-CD3 followed by anti-MGG, andantigen-binding GIF in the supernatant was affinity purified by usingPLA₂ -coupled Sepharose. The preparation could switch the 12H5 cellsfrom the production of glycosylated IgE-BF to the production ofunglycosylated IgE-BF at a dilution of 1:30 to 1:60. BALB/c mice wereimmunized by an i.p. injection of 0.1 ml of the preparation in CFAfollowed by 5 booster injections of the same antigen in incompleteFreund's adjuvant. Two weeks after the last booster injection, theanimals were sacrificed and their spleen cells fused with the B cellline SP 2/0 AG14.

Culture supernatants of hybridomas were tested for the presence of mouseIg. Those hybridomas which produced Ig were further tested for thepresence of anti-GIF by ELISA assays. Maxi-Sorp wells were coated withPLA₂ -binding GIF, and binding of mouse Ig in the culture supernatant tothe GIF-coated wells was determined by using HRP-coupled anti-mouseIgG+A+M. Culture supernatants of 8 hybridomas gave substantialabsorbance by ELISA. The presence of anti-GIF antibody in thesupernatants of the 8 hybridomas was confirmed by incubating an aliquotof affinity-purified PLA₂ -binding GIF with each culture supernatant,followed by filtration of the mixture through Centricon 100 (Amersham).Assay of the filtrates for GIF activity showed that the antibodies fromall 8 of the hybridomas bound GIF, whereas filtrate of PLA₂ -binding GIFitself had the activity.

The same culture supernatants were then examined for the presence ofmonoclonal antibody against nonspecific GIF. Maxi-Sorp plates werecoated with either affinity-purified nonspecific GIF or antigen-specificGIF, and culture supernatant of each B cell hybridoma was added to thewells. After 2 hr incubation at 37° C., the wells were washed and a1:2000 dilution of HRP-coupled goat anti-mouse IgG+A+M was added to eachwell. ELISA signal was developed by peroxidase substrate and measured at405 nm. Absorption of control wells, which were coated with GIF andincubated with HRP-coupled antibodies and substrates, was subtracted.The results of the experiments indicated that only two hybridomas, i.e.,110 and 205, gave substantially higher ELISA signal with PLA₂ -bindingGIF (i.e., antigen specific GIF) than with antigen non specific GIF. Thehybridomas 110 and 205 were subcloned by limiting dilutions, and culturesupernatant of each clone was tested again by ELISA for the selectivebinding of monoclonal antibodies to PLA₂ -binding GIF. After repeatedsubclonings, subclones of each of the two hybridomas, i.e., 110BH3 and205AD2, were obtained whose culture supernatants gave ELISA signals onlywith antigen binding GIF. The monoclonal antibody 110BH3 was μκ isotype,while 205AD2 was γ₁ κ isotype.

Confirmation that both monoclonal antibodies bound PLA₂ -specific GIFbut failed to bind nonspecific GIF was performed in a bioassay. Aliquotsof the GIF preparations were incubated overnight with the culturesupernatant of either 110BH3 cells or 205AD2 cells, and the mixtureswere filtered through Centricon 100. Determination of GIF activity inthe filtrates by using the 12H5 cells showed that both antibodies boundPLA₂ -binding GIF, but failed to bind nonspecific GIF. Next, the mAb110BH3 was enriched from the culture supernatant of the hybridoma byprecipitation with 1/3 saturation of ammonium sulfate, and 5 mg of IgMwere coupled to 1.5 ml of Sepharose. The PLA₂ -binding GIF andnonspecific GIF from the AC5 cells were then fractionated on theantibody-coupled Sepharose. The PLA₂ -binding GIF was prepared fromculture supernatants of anti-CD3-treated AC5 cells byaffinity-purification on PLA₂ -coupled Sepharose, while nonspecific GIFwas prepared from culture supernatants of unstimulated AC5 cells byusing 388F₁ -coupled Sepharose. The results shown in Table XII indicatedthat PLA₂ -binding GIF bound to the immunosorbent and was recovered byelution at acid pH, while nonspecific GIF failed to be retained on theimmunosorbent.

                  TABLE XIII                                                      ______________________________________                                        FRACTIONATION OF GIF PREPARATIONS ON                                          110 BH3-COUPLED SEPHAROSE.sup.a                                               FRACTION FROM                                                                             PLA.sub.2 -BINDING GIF                                                                       NON-SPECIFIC GIF                                   IMMUNOSORBENT.sup.b                                                                       Dilution                                                                              GIF Activity.sup.c                                                                       Dilution                                                                            GIF activity.sup.c                       ______________________________________                                        Unfractionated                                                                            1:10    25/0(+)    1:30  28/0(+)                                  Flow-through                                                                              1:2     0/30(-)    1:30  29/4(+)                                  Wash        1:2     0/23(-)    1:2   3/32(-)                                  Eluate      1:10    25/0(+)    1:2   0/30(-)                                  Medium control      0/33             1/30                                     ______________________________________                                         .sup.a PLA.sub.2binding GIF and nonspecific GIF could switch the 12H5         cells from the formation of glycosylated IgEBF to the formation of            unglycosylated IgEBF at the dilution of 1:10 and 1:30, respectively.          .sup.b One ml of GIF preparation was mixed with 0.25 ml of 110 BH3coupled     Sepharose. Flowthrough fraction and 1 ml washing with DPBS were combined      (Flowthrough fraction). Columns were washed with 5 ml BPBS (wash) and the     eluted with 1 ml glycine HCl buffer, pH 3.0 (Eluate). After dialysis, eac     fraction was concentrated to 1.0 ml for GIF assay.                            .sup.c GIF activity was determined by using 12H5 cells. Numbers in this       column represent percent rosette inhibition by the effluent/eluate            fractions from lentil lectin Sepharose (c.f. method). (+)(-) indicate         presence or absence of GIF activity.                                     

Attempts were made to detect antigen-binding GIF and nonspecific GIF byELISA. Nonspecific GIF was enriched by fractionation of culturesupernatants of unstimulated AC5 cells by ion-exchange columnchromatography on a DEAE-Sepharose column. Flow-through fraction withTris buffer containing 50 mM NaCl was concentrated. The antigen-bindingfactors in the culture supernatants of anti-CD3-stimulated cells werepurified by using PLA₂ -coupled Sepharose, and the factors in acideluates was further fractionated on 110BH3-coupled Sepharose. Bothantigen-specific and nonspecific GIF preparations switched the 12H5cells from the formation of glycosylated IgE-BF to the formation ofunglycosylated IgE-BF at a dilution of 1:60. Next, Max-Sorb wells werecoated with serial dilutions of one of the preparations and, afterblocking, the mAb 388F₁ or 110 BH3 was applied to the wells. mAb 388F₁gave ELISA signals with both nonspecific GIF and antigen-binding GIFpreparations, while mAb 110-BH3 reacted with antigen-binding factor, butnot with the nonspecific GIF. The ELISA signals appear to be due tospecific binding of mAb because no signal was detected when the mAb wasreplaced with irrelevant IgG_(2a) or IgM in the assays.

Since the affinity purified antigen-binding factor described above wasobtained by using PLA₂ -Sepharose followed by 110BH3-Sepharose, and thepreparation was analyzed by SDS-PAGE. Antigen-binding GIF was purifiedby using PLA₂ -coupled Sepharose, followed by 110BH3-coupled Sepharose.Acid eluate fraction from the immunosorbent was dialyzed againstdistilled water in the presence of 0.01% SDS and lyophilized. Thepreparation was analyzed by SDS-PAGE under reducing and non-reducingconditions and silver staining. Under non-reducing conditions, thepreparation gave three major bands of 85 kDa, 66 kDa, 58 kDa and a minorband of 13 kDa. Under reducing conditions, the 85 kDa band disappearedand several new bands were detected. Since one of the major bandsdetected under reducing conditions was 14 kDa, the mobility of whichcorresponded to that of nonspecific GIF, analysis was done to determineif this band was GIF. Another preparation of antigen-binding GIF wasobtained from the culture supernatant of anti-CD3-stimulated AC5 cellsby affinity chromatography on 110BH3-Sepharose. Titration of GIFactivity in serial dilutions of flow-through fraction and acid eluatefractions from the immunosorbent using 12H5 cells showed that themajority of GIF activity in the culture supernatant bound to theimmunosorbent and was recovered by acid elution. Activity was detectedin a 1:30 dilution of the eluate fraction, while the flow-throughfraction gave the GIF titer of 1:6. As shown in Table XIV, the eluatefraction gave ELISA signal with both mAb 110BH3 and 205AD2, while theeffluent fraction failed to give a significant ELISA signal with theantibody. When antibody to nonspecific GIF (388F₁) was employed,however, the flow-through fraction gave a weak but definite ELISAsignal, although the eluate fraction contained much higher concentrationof GIF. The eluate fraction was then analyzed by SDS-PAGE under reducingconditions followed by immunblotting using polyclonal antibodies againstrGIF. Recombinant GIF from E. coli was applied to the next well of thesame gel. After electrophoresis under reducing conditions, proteins inthe gels were transferred to PVDF membranes, which were then treatedwith γ globulin fraction of anti-rGIF. In both membranes, rGIF employedas a control gave a clear band on X-ray film (not shown). Sincemolecular weight markers did not include any protein of less than 18kDa, rGIF band on x-ray film was used as 13 kDa marker. The resultsindicated that the antibodies actually bound to the 13 kDa band. Therelationship between the 13 kDa band and nonspecific GIF was confirmedby analyzing nonspecific GIF, which was purified from culturesupernatant of unstimulated AC5 cells by affinity chromatography on388F₁ -coupled Sepharose. Analysis of the nonspecific GIF preparation bySDS-PAGE and immunoblotting with anti-rGIF antibodies showed that thispreparation also contained a 13 kDa protein which reacted with theantibodies.

                  TABLE XIV                                                       ______________________________________                                        DISTRIBUTION OF GIF ACTIVITY AND GIF ANTIGENS                                 BETWEEN FLOW-THROUGH AND ELUATE FRACTIONS                                     FROM 110BH3-SEPHAROSE.sup.a                                                   FRACTION FROM 110- ELISA SIGNAL                                               BH3-SEPHAROSE                                                                              GIF.sup.b TITER                                                                         205DA2.sup.c                                                                           110BH3.sup.c                                                                         388F.sub.1.sup.d                       ______________________________________                                        Flow-through 1:3       0.063    0.093  0.36                                   Acid-eluate  1:15      0.750    1.068  0.86                                   ______________________________________                                         .sup.a Culture supernatant of antiCD3-treated cells was concentrated and      fractionated on 110BH3Sepharose. Fractions were concentrated to original      volume of the concentrated supernatant, diluted twice and then titrated       for GIF activity and ELISA assays.                                            .sup.b Maximal dilution of the fraction that could switch the 12H5 cells      from the formation of glycosylated IgEBF to the formation of                  unglycosylated IgEBF.                                                         .sup.c Absorption at 405 nm.                                                  .sup.d Absorption at 490 nm.                                             

It was suspected that the 13 kDa GIF and an antigen-binding polypeptidechain were associated with each other to form antigen-binding GIF. Ifthis was the case, the molecular size of antigen-binding GIF would belarger than that of nonspecific GIF under physiological conditions. Totest this possibility, PLA₂ -binding GIF from AC5 cells was partiallypurified by using 110-BH3-Sepharose, and the preparation wasfractionated by gel filtration through a Superose 12 column. Eachfraction was assessed for antigen-binding GIF by ELISA using mAb 388F₁and 110 BH3. The results indicated that the majority of GIF, which wasdetected by mAb 388F₁, was eluted from the column between 55.5 and 60.5min. with a peak at 58.5 min. The size of the molecule, estimated fromits elution time, was 74 kDa. As expected, the fractions contained GIFactivity as determined by bioassay using 12H5 cells. It should be notedthat the GIF-containing fractions gave ELISA signal with mAb 110BH3. Theresults strongly suggest that the antigenic determinant recognized bymAb 388F₁ and that recognized by mAb 110BH3 are associated with the samemolecules.

If antigen-binding GIF actually consists of an antigen-binding chain andnonspecific GIF, the antigen-binding GIF may be dissociated intoseparate polypeptides by reduction and alkylation treatment.

In order to investigate this possibility, affinity-purifiedantigen-binding GIF was reduced in 10 mM DTT. After alkylation withiodoacetamide, the sample was applied to the same Superose 12 column,and the distribution of 388F₁ -antigen and 110BH3-antigen was determinedby ELISA. The results indicated that approximately one half of GIF inthe reduced and alkylated material was recovered in a fraction of whichelution time corresponded to that of 15 kDa molecule. Since the samefraction did not contain GIF when the original antigen-binding GIF wasfractionated, the 15 kDa GIF appears to be derived from theantigen-binding GIF. The experiment also showed two peaks of moleculesrecognized by 110BH3; the first peak corresponded to the originalantigen-binding GIF, while the elution time of the second peakcorresponded to 62-64 kDa. Since the latter fraction did not contain asignificant amount of GIF, as determined by ELISA, the protein in thefraction should represent a cleavage product of antigen-binding GIFresponsible for antigen specific binding.

C. Epitope specificity of antigen-binding GIF

Experiments were carried out to confirm that the PLA₂ -binding GIF isspecific for bee venom PLA₂. The antigen-binding GIF was purified fromculture supernatants of anti-CD3-stimulated AC5 cells by absorption withPLA₂ -coupled Sepharose followed by elution of bound proteins at acidpH. An aliquot of the preparation was mixed overnight with OVA-coupledSepharose, and GIF activity in the flow-through and acid eluatefractions was determined. As expected, essentially all GIF activityfailed to be retained in OVA-Sepharose, and was recovered in aflow-through fraction. The OVA-Sepharose was washed with DPBS, and theimmunosorbent was eluted with glycine-HCl buffer, pH 3.0. However, GIFactivity was not detected in the acid eluate fraction.

Since previous experiments on PLA₂ -binding GIF from the murine Tshybridoma 3B3 have shown that the factor had affinity for the peptiderepresenting amino acid residues 19-34 in bee venom PLA₂ molecules, itwas decided to investigate whether human antigen-binding GIF from AC5cells might bind to Sepharose coupled with the synthetic peptide,representing amino acids 19-35 from PLA₂ molecules. As shown in TableXV, essentially all GIF activity in the preparation was absorbed withthe p19-35-Sepharose, and was recovered by elution at acid pH. In orderto confirm the epitope specificity, aliquots of the PLA₂ -binding GIFwere incubated for 6 hours with 0.2 mg/ml of a synthetic peptide,representing amino acid 13-28, 19-35, or 25-40, and each mixture wasabsorbed with PLA₂ -Sepharose. Determination of GIF activity in theflow-through fraction and acid eluate fraction indicated that thebinding of GIF to PLA₂ -Sepharose was prevented by p13-28 or p19-35, butnot by p25-40 (Table XV). The results were confirmed by ELISA. When thePLA₂ -binding GIF was absorbed with PLA₂ -Sepharose in the presence orabsence of p25-40, acid eluate fraction from the immunosorbent gaveELISA signal with 110BH3, while the flow-through fraction failed to givethe signal. If the same PLA₂ -binding GIF was absorbed with the sameimmunosorbent in the presence of p19-35, the flow-through fraction, butnot the acid eluate fraction, gave ELISA signal with the monoclonalantibody. The results collectively show that the sequence of amino acid19-28 in the PLA₂ molecule contained the epitope which was recognized byantigen-binding GIF.

                  TABLE XV                                                        ______________________________________                                        EPITOPE SPECIFICITY OF PLA.sub.2 -BINDING GIF                                               PEPTIDE                                                                              GIF ACTIVITY.sup.c                                       EXP  IMMUNOSORBENT  ADDED    Flow-through                                                                            Eluate                                 ______________________________________                                        1.sup.a                                                                            PLA.sub.2 -Sepharose                                                                         none     2/28(-)   23/6(+)                                     P19-35-Sepharose                                                                             none     3/30(-)   28/4(+)                                     Medium Control none     4/28      --                                     2.sup.b                                                                            PLA.sub.2 -Sepharose                                                                         none     0/28(-)   27/3(+)                                                    P13-28   22/0(+)   0/23(-)                                                    P19-35   20/5(+)   0/28(-)                                                    P25-40   2/25(-)   23/0 (+)                                    Medium Control none     3/24      --                                     ______________________________________                                         .sup.a 6 ml of acid eluate fraction from PLA.sub.2Sepharose, of which GIF     titer was 1:10, was fractionated on 1.5 ml of Sepharose coupled with          P19-35. Each of the flowthrough, wash and acid eluate fractions were          adjusted to 6.0 ml, and their GIF activity was determined by using 12H5       cells.                                                                        .sup.b A 0.5 ml aliquot of the acid eluate fraction from                      PLA.sub.2Sepharose, of which GIF titer was 1:30, was mixed overnight with     0.5 ml PLA.sub.2Sepharose in the presence or absence of the appropriate       peptide. Both flowthrough and acid eluate fractions from                      PLA.sub.2sepharose were adjusted to 1.0 ml, dialyzed against RPMI 1640        medium, and assessed for GIF activity. One ml of a suspension of 12H5         cells was mixed with an equal volume of a sample to be tested, and            cultured in the presence of IgE.                                              .sup.c Values represent precent rosette inhibition by the effluent/eluate     fractions from lentil lectin Sepharose. (+)(-) indicate the presence or       absence of GIF.                                                          

Deposit of Materials

The following cell lines have been deposited with the American TypeCulture Collection, 1301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________                                        Cell Line ATCC Accession No.                                                                              Deposit Date                                      ______________________________________                                        388F.sub.1                                                                              HB 10472          May 31, 1990                                      AC5       HB 10473          May 31, 1990                                      31E9      HB 11052          June 2, 1992                                      110BH3    Y                 May 14, 1993                                      ______________________________________                                    

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the culturedeposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of a deposited strainis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any cell lines that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the amino acid sequence of peptide AP-1 (page 64,line 16);

Sequence ID No. 2 is the amino acid sequence of peptide AP-23 (page 64,line 17);

Sequence ID No. 3 is the amino acid sequence of peptide AN-4 (page 65,line 2);

Sequence ID No. 4 is the amino acid sequence of peptide AN-5 (page 65,line 3);

Sequence ID No. 5 is the amino acid sequence of peptide AN-7 (page 65,line 5);

Sequence ID No. 6 is the amino acid sequence of peptide T-1 (page 65,line 12);

Sequence ID No. 7 is the amino acid sequence of the N-terminalPVDF-immobilized protein of murine GIF (page 65, line 15);

Sequence ID No. 8 is the nucleotide sequence of a 5'→3' primer used formurine GIF cDNA cloning (page 66, line 1);

Sequence ID No. 9 is the nucleotide sequence of a 5'→3' primer used formurine GIF cDNA cloning (page 66, line 2);

Sequence ID No. 10 is the nucleotide sequence of a 5'→3' primer used forhuman GIF cDNA cloning (page 70, lines 1 and 2);

Sequence ID No. 11 is the nucleotide sequence of a 3'→5' primer used forhuman GIF cDNA cloning (page 70, line 3);

Sequence ID No. 12 is the nucleotide sequence of a 5'→3' primer used togenerate AflII and BamHI restriction sites at both ends of murine GIFcDNA (page 71, lines 5 and 6);

Sequence ID No. 13 is the nucleotide sequence of a 3'→5' primer used togenerate AflII and BamHI restriction sites at both ends of murine GIFcDNA (page 71, line 7);

Sequence ID No. 14 is the nucleotide sequence of a 5'→3' primer used togenerate BglII or KpnI sites on human GIF cDNA (page 76, line 17; page78, line 17);

Sequence ID No. 15 is the nucleotide sequence of a 3'→5' primer used togenerate BglII or KpnI sites on human GIF cDNA (page 76, line 18; page78, line 18);

Sequence ID No. 16 is the nucleotide sequence of a 5'→3' primer having aPstI (page 78, line 12);

Sequence ID No. 17 is the nucleotide sequence of a 3'→5' primer having aPstI (page 78, line 13);

Sequence ID Nos. 18, 20, and 22 are nucleotide sequences of 5'→3'primers for cloning human furin cDNA (page 80, lines 3, 5, and 7);

Sequence ID Nos. 19, 21, and 23 nucleotide sequences of 3'→5' primersfor cloning human furin cDNA (page 80, lines 4, 6, and 8);

Sequence ID No. 24 is the nucleotide sequence of a primer (CT1) forhuman pro-ct (page 83, line 5);

Sequence ID No. 25 is the nucleotide sequence (and deduced amino acidsequence) of a primer (CT2) for human pro-ct (page 83, line 5);

Sequence ID No. 26 is the deduced amino acid sequence for the primer(CT2) for human pro-ct;

Sequence ID No. 27 is the nucleotide sequence (and deduced amino acidsequence) of a primer (CT3) for human pro-ct (page 83, line 5);

Sequence ID No. 28 is the deduced amino acid sequence for the primer(CT3) for human pro-ct;

Sequence ID No. 29 is the nucleotide sequence (and deduced amino acid)of a primer (CT4) for human pro-ct (page 83, line 5);

Sequence ID No. 30 is the deduced amino acid sequence for the primer(CT4) for human pro-ct;

Sequence ID No. 31 is the nucleotide sequence (and deduced amino acid)of a primer (CT5) for human pro-ct (page 83, line 5);

Sequence ID No. 32 is the deduced amino acid sequence for the primer(CT5) for human pro-ct;

Sequence ID No. 33 is the nucleotide sequence of a 5' end primer, G1,for isolation of Fc cDNA (page 84, line 23);

Sequence ID No. 34 is the nucleotide sequence of a 3' end primer, G2,for isolation of Fc cDNA (page 84, line 24);

Sequence ID No. 35 is the nucleotide sequence (and deduced amino acidsequence) for a murine GIF (FIG. 1);

Sequence ID No. 36 is the deduced amino acid sequence for the murine GIF(FIG. 1);

Sequence ID No. 37 is the nucleotide sequence (and deduced amino acidsequence) for a human GIF (FIG. 2); and

Sequence ID No. 38 is the deduced amino acid sequence for the human GIF(FIG. 2).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 38                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: AP-1                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..11                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       IleGlyGlyAlaGlnAsnArgAsnTyrSerLys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: AP-23                                                              (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..20                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuLeuCysGlyLeuLeuSerAspArgLeuHisIleSerProAspArg                              151015                                                                        ValTyrIleAsn                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: AN-4                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AspMetAsnAlaAlaAsnValGlyXaaAsnGlySerThrPheAla                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: AN-5                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..13                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspProCysAlaLeuCysSerLeuHisSerIleGlyLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: AN-7                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspArgLeuHisIleSerProAspArgValTyrIleAsnTyrTyr                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: T-1                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..11                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ProMetPheIleValAsnThrAsnValProArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: N-terminal                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetProMetPheIleValAsnThrAsnValProArgAlaSerVal                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..36                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGCCGATGTTCATCGTAAACACCAACGTGCCCCGC36                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..36                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCCGATGCTGTGCAGGCTGCAGAGCGCGCACGGCTC36                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..60                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AACCTTAAGAAAAACCAAGGAGGTAATAAATAATGCCGATGTTCATCGTAAACACCAACG60                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..42                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CACCCGACCTTGTTGAGGTGGAAGCGGATTATCCCTAGGCAA42                                  (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..60                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AACCTTAAGAAAAACCAAGGAGGTAATAAATAATGCCTATGTTCATCGTGAACACCAATG60                (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..43                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCACCCGACCTTGCCAAGGTGGAAGCGAACTATCCCTAGGCAA43                                 (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCCAGATCTAAGCGGATGCCGATGTTCATCGTAAACACC39                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..32                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CCTTGTTGAGGTGGAAGCGGATTCCATGGCAA32                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..26                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AACTGCAGATGGGCTTCCAAAAGTTC26                                                  (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..32                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GACCTGTCGGGGTCTAGATTCGCCGACGTCCA32                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..33                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AAGAATTCCCCCATGGAGCTGAGGCCCTGGTTG33                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..28                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GTGGTTGTCATAGATGTGCGACAGGTAG28                                                (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..28                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ACACCAACAGTATCTACACGCTGTCCAT28                                                (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..28                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TACCCAAATTACTGACCCGGAAGTACTG28                                                (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..21                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ACTACTCCGCAGATGGGTTTA21                                                       (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..29                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TTTCTGGTCTCGCGGGAGACTCTTAAGAA29                                               (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..28                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GAATTCTGTCATGGGCTTCCAAAAGTTC28                                                (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..30                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CTGGACAGCCCCAGATCCAAGAGATCTAGA30                                              LeuAspSerProArgSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      LeuAspSerProArgSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..30                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CTGGACAGACCCATGTCCAAGAGATCTAGA30                                              LeuAspArgProMetSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      LeuAspArgProMetSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..30                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CTGGACAGACCCAGATCCAAGAGATCTAGA30                                              LeuAspArgProArgSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      LeuAspArgProArgSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..30                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CTGGACAGCCCCATGTCCAAGAGATCTAGA30                                              LeuAspSerProMetSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      LeuAspSerProMetSerLysArgSerArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..25                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CTCTAGAGACAAAACTCACACATGC25                                                   (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..28                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GGCGGCCGCCGCACTCATTTACCCGGAG28                                                (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 635 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: murine GIF                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 82..426                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GGCACGACGTCAGGTCCCTGGCTTGGGTCACACCGCGCTTTGTACCGTCCTCCGGTCCAC60                GCTCGCAGTCTCTCCGCCACCATGCCTATGTTCATCGTGAACACCAATGTT111                        MetProMetPheIleValAsnThrAsnVal                                                1510                                                                          CCCCGCGCCTCCGTGCCAGAGGGGTTTCTGTCGGAGCTCACCCAGCAG159                           ProArgAlaSerValProGluGlyPheLeuSerGluLeuThrGlnGln                              152025                                                                        CTGGCGCAGGCCACCGGCAAGCCCGCACAGTACATCGCAGTGCACGTG207                           LeuAlaGlnAlaThrGlyLysProAlaGlnTyrIleAlaValHisVal                              303540                                                                        GTCCCGGACCAGCTCATGACTTTTAGCGGCACGAACGATCCCTGCGCC255                           ValProAspGlnLeuMetThrPheSerGlyThrAsnAspProCysAla                              455055                                                                        CTCTGCAGCCTGCACAGCATCGGCAAGATCGGTGGTGCCCAGAACCGC303                           LeuCysSerLeuHisSerIleGlyLysIleGlyGlyAlaGlnAsnArg                              606570                                                                        AACTACAGTAAGCTGCTGTGTGGCCTGCTGTCCGATCGCCTGCACATC351                           AsnTyrSerLysLeuLeuCysGlyLeuLeuSerAspArgLeuHisIle                              75808590                                                                      AGCCCGGACCGGGTCTACATCAACTATTACGACATGAACGCTGCCAAC399                           SerProAspArgValTyrIleAsnTyrTyrAspMetAsnAlaAlaAsn                              95100105                                                                      GTGGGCTGGAACGGTTCCACCTTCGCTTGAGTCCTGGCCCCACTTAC446                            ValGlyTrpAsnGlySerThrPheAla                                                   110115                                                                        CTGCACCGCTGTTCTTTGAGCCTCGCCTCTCCACGTAGTGTTCTGTGTTTATCCACCGGT506               AGCGATGCCCACCTTCCAGCCGGGAGAAATAAATGGTTTATAAGAGACCAAAAAAAAAAA566               AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA626               AAAAAAAAA635                                                                  (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      MetProMetPheIleValAsnThrAsnValProArgAlaSerValPro                              151015                                                                        GluGlyPheLeuSerGluLeuThrGlnGlnLeuAlaGlnAlaThrGly                              202530                                                                        LysProAlaGlnTyrIleAlaValHisValValProAspGlnLeuMet                              354045                                                                        ThrPheSerGlyThrAsnAspProCysAlaLeuCysSerLeuHisSer                              505560                                                                        IleGlyLysIleGlyGlyAlaGlnAsnArgAsnTyrSerLysLeuLeu                              65707580                                                                      CysGlyLeuLeuSerAspArgLeuHisIleSerProAspArgValTyr                              859095                                                                        IleAsnTyrTyrAspMetAsnAlaAlaAsnValGlyTrpAsnGlySer                              100105110                                                                     ThrPheAla                                                                     115                                                                           (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 557 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: human GIF cDNA                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 75..419                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CAGGCACGTAGCTCAGCGGCGGCGCGGCGCGTGCGTCTGTGCCTCTGCGCGGGTCTCCTG60                GTCCTTCTGCCATCATGCCGATGTTCATCGTAAACACCAACGTGCCCCGC110                         MetProMetPheIleValAsnThrAsnValProArg                                          1510                                                                          GCCTCCGTGCCGGACGGGTTCCTCTCCGAGCTCACCCAGCAGCTGGCG158                           AlaSerValProAspGlyPheLeuSerGluLeuThrGlnGlnLeuAla                              152025                                                                        CAGGCCACCGGCAAGCCCCCCCAGTACATCGCGGTGCACGTGGTCCCG206                           GlnAlaThrGlyLysProProGlnTyrIleAlaValHisValValPro                              303540                                                                        GACCAGCTCATGGCCTTCGGCGGCTCCAGCGAGCCGTGCGCGCTCTGC254                           AspGlnLeuMetAlaPheGlyGlySerSerGluProCysAlaLeuCys                              45505560                                                                      AGCCTGCACAGCATCGGCAAGATCGGCGGCGCGCAGAACCGCTCCTAC302                           SerLeuHisSerIleGlyLysIleGlyGlyAlaGlnAsnArgSerTyr                              657075                                                                        AGCAAGCTGCTGTGCGGCCTGCTGGCCGAGCGCCTGCGCATCAGCCCG350                           SerLysLeuLeuCysGlyLeuLeuAlaGluArgLeuArgIleSerPro                              808590                                                                        GACAGGGTCTACATCAACTATTACGACATGAACGCGGCCAATGTGGGC398                           AspArgValTyrIleAsnTyrTyrAspMetAsnAlaAlaAsnValGly                              95100105                                                                      TGGAACAACTCCACCTTCGCCTAAGAGCCGCAGGGACCCACGCTGTCTGCG449                        TrpAsnAsnSerThrPheAla                                                         110115                                                                        CTGGCTCCACCCGGGAACCCGCCGCACGCTGTGTTCTAGGCCCGCCCACCCCAACCTTCT509               GGTGGGGAGAAATAAACGGTTTAGAGACTAAAAAAAAAAAAAAAAAAA557                           (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      MetProMetPheIleValAsnThrAsnValProArgAlaSerValPro                              151015                                                                        AspGlyPheLeuSerGluLeuThrGlnGlnLeuAlaGlnAlaThrGly                              202530                                                                        LysProProGlnTyrIleAlaValHisValValProAspGlnLeuMet                              354045                                                                        AlaPheGlyGlySerSerGluProCysAlaLeuCysSerLeuHisSer                              505560                                                                        IleGlyLysIleGlyGlyAlaGlnAsnArgSerTyrSerLysLeuLeu                              65707580                                                                      CysGlyLeuLeuAlaGluArgLeuArgIleSerProAspArgValTyr                              859095                                                                        IleAsnTyrTyrAspMetAsnAlaAlaAsnValGlyTrpAsnAsnSer                              100105110                                                                     ThrPheAla                                                                     115                                                                           __________________________________________________________________________

We claim:
 1. The method of producing a substantially pure, biologicallyactive antigen non-specific glycosylation inhibiting factor (GIF) whichcomprises:culturing a eukaryotic cell transformed with a polynucleotidesequence encoding a fusion polypeptide of the formula:

    R.sub.1 -R.sub.2

wherein R₁ is a pro-region of a precursor of a secretory protein, R₂ isan amino acid sequence as in SEQ ID NO:36 or SEQ ID NO:38, the carboxyterminus of the pro-region contains or is modified to contain thesequence ((X₁ X₂)_(n) -Lys-Arg), wherein n is either 1 or 2, X₁ is Lysor Arg, and X₂ is any amino acid; and isolating substantially pureantigen non-specific GIF.
 2. The method of claim 1, wherein thepro-region is calcitonin precursor.
 3. A substantially pure fusionpolypeptide of the formula:

    R.sub.1 -R.sub.2

wherein R₁ is a pro-region of a precursor of a secretory protein, R₂ isan amino acid sequence as in SEQ ID NO:36 or SEQ ID NO:38, and thecarboxy terminus of the pro-region contains or is modified to containthe sequence (X₁ X₂)_(n) -Lys-Arg), wherein n is either 1 or 2, X₁ isLys or Arg, and X₂ is any amino acid.
 4. The fusion polypeptide of claim3, wherein the pro-region is calcitonin precursor.
 5. An isolatedpolynucleotide sequence which encodes a substantially pure fusionpolypeptide of the formula:

    R.sub.1 -R.sub.2

wherein R₁ is a pro-region of a precursor of a secretory protein, R₂ isan amino acid sequence as in SEQ ID NO:36 or SEQ ID NO:38, and thecarboxy terminus of the pro-region contains or is modified to containthe sequence ((X₁ X₂)_(n) -Lys-Arg), wherein n is either 1 or 2, X₁ isLys or Arg, and X₂ is any amino acid.
 6. A recombinant expression vectorcontaining the polynucleotide of claim
 5. 7. The vector of claim 6,wherein the vector is a virus.
 8. The vector of claim 6, wherein thevector is a plasmid.
 9. A host cell containing the vector of claim 6.10. The host cell of claim 9, wherein the host cell is a eukaryote cell.11. The host cell of claim 10, wherein the host cell is a COS-1 cell.12. An isolated polynucleotide sequence encoding antigen non-specificGIF polypeptide as in SEQ ID NO:36 or SEQ ID NO:38.
 13. Thepolynucleotide of claim 12, wherein the GIF sequence is selected fromthe group consisting of SEQ ID NO:35 and SEQ ID NO:37.
 14. A recombinantvector containing the polynucleotide sequence of claim
 12. 15. Thevector of claim 14, wherein the vector is a virus.
 16. The vector ofclaim 14, wherein the vector is a plasmid.
 17. A host cell containingthe vector of claim
 14. 18. The host cell of claim 17, wherein the hostcell is a prokaryote cell.
 19. The host cell of claim 18, wherein thehost cell is E.coli.
 20. The host cell of claim 17, wherein the hostcell is a eukaryote cell.
 21. The host cell of claim 20, wherein thehost cell is a COS-1 cell.
 22. A method of producing biologically activeGIF which comprises:a) culturing a host cell of claim 17; and b)isolating substantially pure GIF from the culture.
 23. The method ofclaim 22, wherein the host cell is a eukaryote cell.
 24. The method ofclaim 23, wherein the host cell is a COS-1 cell.
 25. The method of claim22, wherein the host cell is a prokaryote cell.
 26. The method of claim25, wherein the prokaryote cell is E. coli.